Materials and methods for treatment of autosomal dominant cone-rod dystrophy

ABSTRACT

The present application provides materials and methods for treating a patient with autosomal dominant CORD, both ex vivo and in vivo; materials and methods for editing a GUCY2D gene in a human cell; and materials and methods for editing a R838H, R838C, or R838S mutation in a GUCY2D gene in a human cell. The present application also provides one or more gRNAs or sgRNAs for editing a GUCY2D gene; one or more gRNAs or sgRNAs for editing a R838H, R838C, or R838S mutation in a GUCY2D gene; and a therapeutic comprising at least one or more gRNAs or sgRNAs for editing a R838H, R838C, or R838S mutation in a GUCY2D gene. The present application provides a therapeutic for treating a patient with autosomal dominant CORD. The present application also provides a kit for treating a patient with autosomal dominant CORD. In addition, the present application provides a self-inactivating CRISPR-Cas system.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/899,717 filed Jun. 12, 2020, which is a continuation of InternationalApplication No. PCT/IB2018/060138, filed Dec. 14, 2018, which claims thebenefit of U.S. Provisional Application No. 62/598,682 filed Dec. 14,2017; U.S. Provisional Application No. 62/670,378 filed May 11, 2018;U.S. Provisional Application No. 62/675,306 filed May 23, 2018; and U.S.Provisional Application No. 62/693,100 filed Jul. 2, 2018. The entirecontents of these applications are incorporated herein by reference intheir entirety.

FIELD

The present application provides materials and methods for treatingautosomal dominant Cone-Rod Dystrophy (CORD).

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format via EFS-Web, and is herebyincorporated by reference in its entirety. Said ASCII copy, created onMar. 29, 2021, is named CB2A_Seqlisting.txt and is 10,212,790 bytes insize.

BACKGROUND

Cone-rod dystrophies (CORD) are genetic ocular disorders characterizedby the loss of cone cells, the photoreceptors responsible for bothcentral and color vision. CORD can cause a variety of symptoms includingdecreased central visual acuity and photophobia, which is a reducedability to see colors and an increased sensitivity to light, both ofwhich can be early symptoms. Later symptoms can include night blindnessand further decrease of visual acuity. Mutations associated with CORDhave been identified, including mutations in the GUCY2D gene.

Currently, there are no adequate treatments or therapies to prevent thedevelopment of CORD or to restore vision, and there remains a criticalneed for developing safe and effective treatments for CORD.

SUMMARY

The present disclosure presents a novel method to ameliorate, if noteliminate, an autosomal dominant CORD. The novel approach targets amutation in the GUCY2D gene, such as an R838H, R838C, or R838S mutation,with a method resulting in the reduction or elimination of expression ofthe defective protein encoded by a gene containing the mutation.Furthermore, in some cases, the treatment can be effected with a smallnumber of treatments and, in some cases, with a single treatment. Theresulting therapy can ameliorate autosomal dominant CORD associated witha mutant GUCY2D gene or, in some cases, can eliminate autosomal dominantCORD associated with a mutant GUCY2D gene.

Provided herein is a method for editing a GUCY2D gene in a human cell.The method comprises introducing into the human cell one or more DNAendonucleases to effect one or more SSBs or DSBs within or near theGUCY2D gene or other DNA sequences that encode regulatory elements ofthe GUCY2D gene that results in a deletion, insertion, or correctionthereby creating an edited human cell.

Also provided herein is a method for editing a R838H, R838C, or R838Smutation in a GUCY2D gene in a human cell. The method comprises:introducing into the human cell one or more DNA endonucleases to effectone or more SSBs or DSBs within or near the R838H, R838C, or R838Smutation in a GUCY2D gene that results in a deletion, insertion, orcorrection thereby creating an edited human cell.

Also provided herein is a method for editing a GUCY2D gene in a humancell. The method comprises introducing into the human cell one or moredeoxyribonucleic acid (DNA) endonucleases to effect one or moresingle-strand breaks (SSBs) or double-strand breaks (DSBs) within ornear the GUCY2D gene or other DNA sequences that encode regulatoryelements of the GUCY2D gene that results in a modulation of expressionor function of the GUCY2D gene thereby creating an edited human cell.

Also provided herein is a method for editing a R838H, R838C, or R838Smutation in a GUCY2D gene in a human cell. The method comprises:introducing into the human cell one or more DNA endonucleases to effectone or more SSBs or DSBs within or near the R838H, R838C, or R838Smutation in a GUCY2D gene that results in a modulation of expression orfunction of the GUCY2D gene thereby creating an edited human cell.

Also provided herein is an in vivo method for treating a patient withautosomal dominant CORD. The method comprises: editing a R838H, R838C,or R838S mutation in a GUCY2D gene in a cell of the patient.

Also provided herein is one or more gRNAs for editing a R838H, R838C, orR838S mutation in a GUCY2D gene in a cell from a patient with autosomaldominant CORD. The one or more gRNAs comprises a spacer sequenceselected from the group consisting of nucleic acid sequences in SEQ IDNOs: 5282-5313, 5398-5409, and 5434-5443 of the Sequence Listing.

Also provided herein is a gRNA for editing a R838H or R838C mutation ina GUCY2D gene in a cell from a patient with autosomal dominant CORD. ThegRNA comprises a spacer sequence selected from the group consisting ofnucleic acid sequences in 5398-5409 of the Sequence Listing.

Also provided herein is a gRNA for editing a R838H or R838S mutation ina GUCY2D gene in a cell from a patient with autosomal dominant CORD. ThegRNA comprises a spacer sequence selected from the group consisting ofnucleic acid sequences in 5434-5443 of the Sequence Listing.

Also provided herein is a therapeutic for treating a patient withautosomal dominant Cone-Rod Dystrophy, the therapeutic comprising atleast one or more gRNAs for editing a R838H, R838C, or R838S mutation ina GUCY2D gene. The one or more gRNAs comprises a spacer sequenceselected from the group consisting of nucleic acid sequences in SEQ IDNOs: 5282-5313, 5398-5409, and 5434-5443 of the Sequence Listing.

Also provided herein is a therapeutic for treating a patient withautosomal dominant Cone-Rod Dystrophy, the therapeutic comprising a gRNAfor editing a R838H or R838C mutation in a GUCY2D gene. The gRNAcomprises a spacer sequence selected from the group consisting ofnucleic acid sequences in 5398-5409 of the Sequence Listing.

Also provided herein is a therapeutic for treating a patient withautosomal dominant Cone-Rod Dystrophy, the therapeutic comprising a gRNAfor editing a R838H or R838S mutation in a GUCY2D gene. The gRNAcomprises a spacer sequence selected from the group consisting ofnucleic acid sequences in 5434-5443 of the Sequence Listing.

Also provided herein is a therapeutic for treating a patient withautosomal dominant CORD, the therapeutic formed by a method comprising:introducing one or more DNA endonucleases; introducing one or more gRNAor one or more sgRNA for editing a R838H, R838C, or R838S mutation in aGUCY2D gene; and optionally introducing one or more donor template. Theone or more gRNAs or sgRNAs comprises a spacer sequence selected fromthe group consisting of nucleic acid sequences in SEQ ID NOs: 5282-5313,5398-5409, and 5434-5443 of the Sequence Listing.

Also provided herein is a therapeutic for treating a patient withautosomal dominant CORD, the therapeutic formed by a method comprising:introducing one or more DNA endonucleases; introducing a gRNA or sgRNAfor editing a R838H or R838C mutation in a GUCY2D gene; and optionallyintroducing one or more donor template. The gRNA or sgRNA comprises aspacer sequence selected from the group consisting of nucleic acidsequences in SEQ ID NOs: 5398-5409 of the Sequence Listing.

Also provided herein is a therapeutic for treating a patient withautosomal dominant CORD, the therapeutic formed by a method comprising:introducing one or more DNA endonucleases; introducing a gRNA or sgRNAfor editing a R838H or R838S mutation in a GUCY2D gene; and optionallyintroducing one or more donor template. The gRNA or sgRNA comprises aspacer sequence selected from the group consisting of nucleic acidsequences in SEQ ID NOs: 5434-5443 of the Sequence Listing.

Also provided herein is a kit for treating a patient with autosomaldominant CORD in vivo. The kit comprises one or more gRNAs or sgRNAs forediting a R838H, R838C, or R838S mutation in a GUCY2D gene, one or moreDNA endonucleases; and optionally, one or more donor template. The oneor more gRNAs or sgRNAs comprises a spacer sequence selected from thegroup consisting of nucleic acid sequences in SEQ ID NOs: 5282-5313,5398-5409, and 5434-5443 of the Sequence Listing.

Also provided herein is a kit for treating a patient with autosomaldominant CORD in vivo. The kit comprises a gRNA or sgRNA for editing aR838H or R838C mutation in a GUCY2D gene, one or more DNA endonucleases;and optionally, one or more donor template. The gRNA or sgRNA comprisesa spacer sequence selected from the group consisting of nucleic acidsequences in SEQ ID NOs: 5398-5409 of the Sequence Listing.

Also provided herein is a kit for treating a patient with autosomaldominant CORD in vivo. The kit comprises a gRNA or sgRNA for editing aR838H or R838S mutation in a GUCY2D gene, one or more DNA endonucleases;and optionally, one or more donor template. The gRNA or sgRNA comprisesa spacer sequence selected from the group consisting of nucleic acidsequences in SEQ ID NOs: 5434-5443 of the Sequence Listing.

Also provided herein is a single-molecule guide RNA (sgRNA) comprisingthe nucleic acid sequence of SEQ ID NO: 5285.

Also provided herein is a single-molecule guide RNA (sgRNA) comprisingthe nucleic acid sequence of SEQ ID NO: 5398.

Also provided herein is a single-molecule guide RNA (sgRNA) comprisingthe nucleic acid sequence of SEQ ID NO: 5286.

Also provided herein is a single-molecule guide RNA (sgRNA) comprisingthe nucleic acid sequence of SEQ ID NO: 5464.

Also provided herein is a single-molecule guide RNA (sgRNA) comprisingthe nucleic acid sequence of SEQ ID NO: 5465.

Also provided herein is a single-molecule guide RNA (sgRNA) comprisingthe nucleic acid sequence of SEQ ID NO: 5466.

Also provided herein is a method for editing an R838H mutation within aGUCY2D gene, the method comprising administering a gRNA or sgRNAcomprising SEQ ID NO: 5285.

Also provided herein is a method for editing an R838H mutation within aGUCY2D gene, the method comprising administering a gRNA or sgRNAcomprising SEQ ID NO: 5286.

Also provided herein is a method for editing an R838H mutation or R838Cmutation within a GUCY2D gene, the method comprising administering agRNA or sgRNA comprising SEQ ID NO: 5398.

Also provided herein is a method for editing an R838H mutation within aGUCY2D gene, the method comprising administering a gRNA or sgRNAcomprising SEQ ID NO: 5464.

Also provided herein is a method for editing an R838H mutation within aGUCY2D gene, the method comprising administering a gRNA or sgRNAcomprising SEQ ID NO: 5465.

Also provided herein is a method for editing an R838H mutation or R838Cmutation within a GUCY2D gene, the method comprising administering agRNA or sgRNA comprising SEQ ID NO: 5466.

Also provided herein is a method for treating a patient with an R838Hmutation within a GUCY2D gene, the method comprising administering agRNA or sgRNA comprising SEQ ID NO: 5285 to the patient.

Also provided herein is a method for treating a patient with an R838Hmutation within a GUCY2D gene, the method comprising administering agRNA or sgRNA comprising SEQ ID NO: 5286 to the patient.

Also provided herein is a method for treating a patient with an R838Hmutation or R838C mutation within a GUCY2D gene, the method comprisingadministering a gRNA or sgRNA comprising SEQ ID NO: 5398 to the patient.

Also provided herein is a method for treating a patient with an R838Hmutation within a GUCY2D gene, the method comprising administering agRNA or sgRNA comprising SEQ ID NO: 5464 to the patient.

Also provided herein is a method for treating a patient with an R838Hmutation within a GUCY2D gene, the method comprising administering agRNA or sgRNA comprising SEQ ID NO: 5465 to the patient.

Also provided herein is a method for treating a patient with an R838Hmutation or R838C mutation within a GUCY2D gene, the method comprisingadministering a gRNA or sgRNA comprising SEQ ID NO: 5466 to the patient.

Also provided herein is a self-inactivating CRISPR-Cas system. Theself-inactivating CRISPR-Cas system comprises a first segment, a secondsegment, and one or more third segments. The first segment comprises anucleotide sequence that encodes a polypeptide inducing site-directedmutagenesis. The second segment comprises a nucleotide sequence thatencodes a guide RNA (gRNA) or a single-molecule guide RNA (sgRNA)wherein the gRNA or sgRNA comprise SEQ ID NO: 5285 or 5464. The one ormore third segments comprise a self-inactivating (SIN) site. The gRNA orsgRNA is substantially complementary to the SIN site. The gRNA or sgRNAis substantially complementary to a genomic target sequence.

Also provided herein is a self-inactivating CRISPR-Cas system. Theself-inactivating CRISPR-Cas system comprises a first segment, a secondsegment, and one or more third segments. The first segment comprises anucleotide sequence that encodes a polypeptide inducing site-directedmutagenesis. The second segment comprises a nucleotide sequence thatencodes a guide RNA (gRNA) or a single-molecule guide RNA (sgRNA)wherein the gRNA or sgRNA comprise SEQ ID NO: 5398 or 5466. The one ormore third segments comprise a self-inactivating (SIN) site. The gRNA orsgRNA is substantially complementary to the SIN site. The gRNA or sgRNAis substantially complementary to a genomic target sequence.

Also provided herein is a self-inactivating CRISPR-Cas system. Theself-inactivating CRISPR-Cas system comprises a first segment, a secondsegment, and one or more third segments. The first segment comprises anucleotide sequence that encodes a SpCas9 or any variants thereof. Thesecond segment comprises a nucleotide sequence that encodes a gRNA orsgRNA. The one or more third segments comprise a self-inactivating (SIN)site. The gRNA or sgRNA is substantially complementary to the SIN site.The gRNA or sgRNA is substantially complementary to a genomic targetsequence. The SIN site comprises a sequence selected from the groupconsisting of SEQ ID NOs: 5478-5492

It is understood that the inventions described in this specification arenot limited to the examples summarized in this Summary. Various otheraspects are described and exemplified herein.

BRIEF DESCRIPTION OF THE DRAWINGS

Various aspects of materials and methods for treatment of automosomaldominant CORD disclosed and described in this specification can bebetter understood by reference to the accompanying figures, in which:

FIGS. 1A-B depict the type II CRISPR/Cas system.

FIG. 1A depicts the type II CRISPR/Cas system including gRNA.

FIG. 1B depicts the type II CRISPR/Cas system including sgRNA.

FIGS. 2A-C show the sgRNA sequence, the target DNA sequence, and thereverse strand of the target DNA sequence to which the sgRNA binds, foreach of 42 sgRNA sequences.

FIG. 2A shows the sgRNA sequence, for each of 42 sgRNA sequences.

FIG. 2B shows the target DNA sequence, for each of 42 sgRNA sequences.

FIG. 2C shows the reverse strand of the target DNA sequence to which thesgRNA binds, for each of 42 sgRNA sequences.

FIGS. 2D-F show the sgRNA sequence, the target DNA sequence, and thereverse strand of the target DNA sequence to which the sgRNA binds, foreach of 22 sgRNA sequences.

FIG. 2D shows the sgRNA sequence, for each of 22 sgRNA sequences.

FIG. 2E shows the target DNA sequence, for each of 22 sgRNA sequences.

FIG. 2F shows the reverse strand of the target DNA sequence to which thesgRNA binds, for each of 22 sgRNA sequences.

FIGS. 3A-F are maps depicting the target DNA sequences for gRNAstargeting the wild-type GUCY2D gene or gRNAs targeting the R838H, R838C,or R838S mutation within the GUCY2D gene.

FIG. 3A is a map depicting the target DNA sequences for gRNAs thattarget the wild-type GUCY2D gene.

FIG. 3B is a map depicting the target DNA sequences for gRNAs thattarget the R838H mutation within the GUCY2D gene.

FIG. 3C is a map depicting the target DNA sequences for gRNAs thattarget the R838C mutation within the GUCY2D gene.

FIG. 3D is a map depicting the target DNA sequences for gRNAs thattarget the R838S mutation within the GUCY2D gene.

FIG. 3E is a map depicting the target DNA sequences for gRNAs thattarget both the R838H mutation and R838C mutation within the GUCY2Dgene.

FIG. 3F is a map depicting the target DNA sequences for gRNAs thattarget both the R838H mutation and R838S mutation within the GUCY2Dgene.

FIGS. 4A-E describe the on-target editing efficiency of sgRNAs targetingthe wild-type GUCY2D gene and the off-target editing efficiency ofsgRNAs targeting the R838H, R838C, or R838S mutation within the GUCY2Dgene.

FIG. 4A describes the on-target editing efficiency of sgRNAs targetingthe wild-type GUCY2D gene (sgRNAs comprising SEQ ID NO: 5274 or 5278)and the off-target editing efficiency of sgRNAs targeting the R838H,R838C, or R838S mutation within the GUCY2D gene (sgRNAs comprising SEQID NO: 5284, 5289, 5296, 5300, 5304, 5308, 5398, or 5403).

FIG. 4B describes the on-target editing efficiency of sgRNAs targetingthe wild-type GUCY2D gene (sgRNAs comprising SEQ ID NO: 5275 or 5279)and the off-target editing efficiency of sgRNAs targeting the R838H,R838C, or R838S mutation within the GUCY2D gene (sgRNAs comprising SEQID NO: 5285, 5290, 5297, 5301, 5305, 5309, 5399, or 5404).

FIG. 4C describes the on-target editing efficiency of gRNAs targetingthe wild-type GUCY2D gene (sgRNAs comprising SEQ ID NO: 5276 or 5280)and the off-target editing efficiency of sgRNAs targeting the R838H,R838C, or R838S mutation within the GUCY2D gene (sgRNAs comprising SEQID NO: 5286, 5291, 5298, 5302, 5306, 5310, 5400, or 5405).

FIG. 4D describes the on-target editing efficiency of sgRNAs targetingthe wild-type GUCY2D gene (sgRNAs comprising SEQ ID NO: 5277 or 5281)and the off-target editing efficiency of sgRNAs targeting the R838H,R838C, or R838S mutation within the GUCY2D gene (sgRNAs comprising SEQID NO: 5287, 5292, 5299, 5303, 5307, 5311, 5402, or 5407).

FIG. 4E describes the off-target editing efficiency of sgRNAs targetingthe R838H or R838C mutation within the GUCY2D gene (sgRNAs comprisingSEQ ID NO: 5288, 5293, 5401, or 5406).

FIG. 5 describes the on-target editing efficiency of sgRNAs targetingthe wild-type GUCY2D gene of a plasmid (a sgRNA comprising SEQ ID NO:5274) and the on-target editing efficiency of sgRNAs targeting the R838Hwithin the GUCY2D gene of a plasmid (sgRNAs comprising SEQ ID NO: 5284,5285, 5286, 5287, 5288, 5398, 5399, 5400, 5401, or 5402).

FIGS. 6A-B describe the on-target and off-target editing efficiency of aplasmid transcribed sgRNA targeting the wild-type GUCY2D gene (a sgRNAcomprising SEQ ID NO: 5274) at 48 or 72 hours post transfection and theon-target and off-target editing efficiency of plasmid transcribedsgRNAs targeting the R838H or R838C mutation within the GUCY2D gene(sgRNAs comprising SEQ ID NO: 5285, 5286, 5398, 5399, or 5402) at 48 or72 hours post transfection.

FIG. 6A describes the on-target and off-target editing efficiency of aplasmid transcribed sgRNA targeting the wild-type GUCY2D gene (a sgRNAcomprising SEQ ID NO: 5274) at 48 hours post transfection and theon-target and off-target editing efficiency of plasmid transcribedsgRNAs targeting the R838H or R838C mutation within the GUCY2D gene(sgRNAs comprising SEQ ID NO: 5285, 5286, 5398, 5399, or 5402) at 48hours post transfection.

FIG. 6B describes the on-target and off-target editing efficiency of aplasmid transcribed sgRNA targeting the wild-type GUCY2D gene (a sgRNAcomprising SEQ ID NO: 5274) at 72 hours post transfection and theon-target and off-target editing efficiency of plasmid transcribedsgRNAs targeting the R838H or R838C mutation within the GUCY2D gene of aplasmid (sgRNAs comprising SEQ ID NO: 5285, 5286, 5398, 5399, or 5402)at 72 hours post transfection.

FIGS. 7A-J show a donor plasmid used to prepare 3 different HEK 293FTreporter cell lines and flow cytometry data for the 3 different HEK293FT reporter cell lines that are co-transfected with a plasmidencoding SpCas9 driven by human EF1α core promoter and a plasmidcontaining R838H_Sp_T2 sgRNA (sgRNA comprising SEQ ID NO: 5285), orR838CH_Sp_T1 sgRNA (sgRNA comprising SEQ ID NO: 5398) under U6 promoter,or no sgRNA.

FIG. 7A depicts a donor plasmid comprising a Cas9 target site (either awild-type GUCY2D gene, a GUCY2D gene comprising a R838H mutation, or aGUCY2D gene comprising a R838C mutation) fused to a blue flurescenceprotein.

FIG. 7B shows flow cytometry data for HEK 293FT reporter cells that havethe wild-type GUCY2D gene as the Cas9 target site and that areco-transfected with a plasmid encoding SpCas9 driven by human elongationfactor 1 alpha (EF1α) core promoter and a plasmid containing R838H_Sp_T2sgRNA (sgRNA comprising SEQ ID NO: 5285) under U6 promoter.

FIG. 7C shows flow cytometry data for HEK 293FT reporter cells that havethe wild-type GUCY2D gene as the Cas9 target site and that areco-transfected with a plasmid encoding SpCas9 driven by human EF1α corepromoter and a plasmid containing R838CH_Sp_T1 sgRNA (sgRNA comprisingSEQ ID NO: 5398) under U6 promoter.

FIG. 7D shows flow cytometry data for HEK 293FT reporter cells that havethe wild-type GUCY2D gene as the Cas9 target site and that are nottransfected with any sgRNA.

FIG. 7E shows flow cytometry data for HEK 293FT reporter cells that havethe R838H mutation as the Cas9 target site and that are co-transfectedwith a plasmid encoding SpCas9 driven by human EF1α core promoter and aplasmid containing R838H_Sp_T2 sgRNA (sgRNA comprising SEQ ID NO: 5285)under U6 promoter.

FIG. 7F shows flow cytometry data for HEK 293FT reporter cells that havethe R838H mutation as the Cas9 target site and that are co-transfectedwith a plasmid encoding SpCas9 driven by human EF1α core promoter and aplasmid containing R838CH_Sp_T1 sgRNA (sgRNA comprising SEQ ID NO: 5398)under U6 promoter.

FIG. 7G shows flow cytometry data for HEK 293FT reporter cells that havethe R838H mutation as the Cas9 target site and that are not transfectedwith any sgRNA.

FIG. 7H shows flow cytometry data for HEK 293FT reporter cells that havethe R838C mutation as the Cas9 target site and that are co-transfectedwith a plasmid encoding SpCas9 driven by human EF1α core promoter and aplasmid containing R838H_Sp_T2 sgRNA (sgRNA comprising SEQ ID NO: 5285)under U6 promoter.

FIG. 7I shows flow cytometry data for HEK 293FT reporter cells that havethe R838C mutation as the Cas9 target site and that are co-transfectedwith a plasmid encoding SpCas9 driven by human EF1α core promoter and aplasmid containing R838CH_Sp_T1 sgRNA (sgRNA comprising SEQ ID NO: 5398)under U6 promoter.

FIG. 7J shows flow cytometry data for HEK 293FT reporter cells that havethe R838C mutation as the Cas9 target site and that are not transfectedwith any sgRNA.

FIGS. 8A-B are graphs showing the percent reduction of cGMP and absolutecGMP for HEK293T-SpCas9 cells co-transfected with a vector containingR838H cDNA and IVT sgRNAs that target the R838H mutation within theGUCY2D gene.

FIG. 8A is a graph showing the percent reduction of cGMP forHEK293T-SpCas9 cells co-transfected with a vector containing R838H cDNAand IVT sgRNAs that target the R838H mutation within the GUCY2D gene(sgRNAs comprising SEQ ID NO: 5284, 5285, 5286, 5287, 5288, 5289, 5290,5291, 5292, or 5293).

FIG. 8B is a graph showing the absolute cGMP for HEK293T-SpCas9 cellsco-transfected with a vector containing R838H cDNA and IVT sgRNAs thattarget the R838H mutation within the GUCY2D gene (sgRNAs comprising SEQID NO: 5284, 5285, 5286, 5287, 5288, 5289, 5290, 5291, 5292, or 5293).

FIGS. 9A-B are graphs showing the percent reduction of cGMP and absolutecGMP for HEK293T-SpCas9 cells co-transfected with a vector containingR838H cDNA and IVT sgRNAs that target the R838H mutation within theGUCY2D gene.

FIG. 9A is a graph showing the percent reduction of cGMP forHEK293T-SpCas9 cells co-transfected with a vector containing R838H cDNAand IVT sgRNAs that target the R838H mutation within the GUCY2D gene(sgRNAs comprising SEQ ID NO: 5285, 5286, or 5291).

FIG. 9B is a graph showing the absolute cGMP for HEK293T-SpCas9 cellsco-transfected with a vector containing R838H cDNA and IVT sgRNAs thattarget the R838H mutation within the GUCY2D gene (sgRNAs comprising SEQID NO: 5285, 5286, or 5291).

FIGS. 10A-B are graphs showing the percent reduction of cGMP andabsolute cGMP for HEK293T-SpCas9 cells co-transfected with a vectorcontaining R838H cDNA and pAAV-U6-R838 sgRNA that targets the R838Hmutation within the GUCY2D gene.

FIG. 10A is a graph showing the percent reduction of cGMP forHEK293T-SpCas9 cells co-transfected with a vector containing R838H cDNAand pAAV-U6-R838 sgRNA that targets the R838H mutation within the GUCY2Dgene (pAAV-5285 or pAAV-5286).

FIG. 10B is a graph showing the absolute cGMP for HEK293T-SpCas9 cellsco-transfected with a vector containing R838H cDNA and pAAV-U6-R838sgRNA that targets the R838H mutation within the GUCY2D gene (pAAV-5285or pAAV-5286).

FIGS. 11A-D depict the structural arrangement of SIN-AAV SpCas9 version1 (sEF1α promoter), SIN-AAV SpCas9 version 2 (sEF1α promoter),Non-SIN-AAV SpCas9 (sEF1α promoter), and the AAV sequence of pSIA012 andpSIA015.

FIG. 11A depicts the structural arrangement of SIN-AAV SpCas9 version1(sEF1α promoter).

FIG. 11B depicts the structural arrangement of SIN-AAV SpCas9 version 2(sEF1α promoter).

FIG. 11C depicts the structural arrangement of Non-SIN-AAV SpCas9 (sEF1αpromoter).

FIG. 11D depicts the structural arrangement of an AAV sequence ofpSIA012 and pSIA015. pSIA012 is a plasmid comprising an AAV sequencethat encodes a sgRNA comprising SEQ ID NO: 5285. pSIA015 is a plasmidcomprising an AAV sequence that encodes a sgRNA comprising SEQ ID NO:5398.

FIGS. 12A-U show flow cytometry data for 3 different HEK 293FT reportercell lines that are co-transfected with pSIA012, a plasmid comprising anAAV sequence that encodes R838H_Sp_T2 sgRNA (a sgRNA comprising SEQ IDNO: 5285) or pSIA015, a plasmid comprising an AAV sequence that encodesR838CH_Sp_T1 sgRNA (a sgRNA comprising SEQ ID NO: 5398), and either (1)a SIN-AAV SpCas9 version 1 (sEF1α promoter), (2) a SIN-AAV SpCas9version 2 (sEF1α promoter), or (3) a Non-SIN-AAV SpCas9 (sEF1αpromoter).

FIG. 12A shows flow cytometry data for HEK 293FT reporter cells thathave the wild-type GUCY2D gene as the Cas9 target site and that areco-transfected with (1) pSIA012 and (2) a SIN-AAV SpCas9 version 1(sEF1α).

FIG. 12B shows flow cytometry data for HEK 293FT reporter cells thathave the wild-type GUCY2D gene as the Cas9 target site and that areco-transfected with (1) pSIA015 and (2) a SIN-AAV SpCas9 version 1(sEF1α).

FIG. 12C shows flow cytometry data for HEK 293FT reporter cells thathave the R838H mutation as the Cas9 target site and that areco-transfected with (1) pSIA012 and (2) a SIN-AAV SpCas9 version 1(sEF1α).

FIG. 12D shows flow cytometry data for HEK 293FT reporter cells thathave the R838H mutation as the Cas9 target site and that areco-transfected with (1) pSIA015 and (2) a SIN-AAV SpCas9 version 1(sEF1α).

FIG. 12E shows flow cytometry data for HEK 293FT reporter cells thathave the R838C mutation as the Cas9 target site and that areco-transfected with (1) pSIA012 and (2) a SIN-AAV SpCas9 version 1(sEF1α).

FIG. 12F shows flow cytometry data for HEK 293FT reporter cells thathave the R838C mutation as the Cas9 target site and that areco-transfected with (1) pSIA015 and (2) a SIN-AAV SpCas9 version 1(sEF1α).

FIG. 12G shows flow cytometry data for HEK 293FT reporter cells thathave the wild-type GUCY2D gene as the Cas9 target site and that areco-transfected with (1) pSIA012 and (2) a SIN-AAV SpCas9 version 2(sEF1α).

FIG. 12H shows flow cytometry data for HEK 293FT reporter cells thathave the wild-type GUCY2D gene as the Cas9 target site and that areco-transfected with a (1) pSIA015 and a (2) SIN-AAV SpCas9 version 2(sEF1α).

FIG. 12I shows flow cytometry data for HEK 293FT reporter cells thathave the R838H mutation as the Cas9 target site and that areco-transfected with (1) pSIA012 and (2) a SIN-AAV SpCas9 version 2(sEF1α).

FIG. 12J shows flow cytometry data for HEK 293FT reporter cells thathave the R838H mutation as the Cas9 target site and that areco-transfected with (1) pSIA015 and (2) a SIN-AAV SpCas9 version 2(sEF1α).

FIG. 12K shows flow cytometry data for HEK 293FT reporter cells thathave the R838C mutation as the Cas9 target site and that areco-transfected with (1) pSIA012 and (2) a SIN-AAV SpCas9 version 2(sEF1α).

FIG. 12L shows flow cytometry data for HEK 293FT reporter cells thathave the R838C mutation as the Cas9 target site and that areco-transfected with (1) pSIA015 and (2) a SIN-AAV SpCas9 version 2(sEF1α).

FIG. 12M shows flow cytometry data for HEK 293FT reporter cells thathave the wild-type GUCY2D gene as the Cas9 target site and that areco-transfected with (1) pSIA012 and (2) a Non-SIN-AAV SpCas9 (sEF1α).

FIG. 12N shows flow cytometry data for HEK 293FT reporter cells thathave the wild-type GUCY2D gene as the Cas9 target site and that areco-transfected with (1) pSIA015 and (2) a Non-SIN-AAV SpCas9 (sEF1α).

FIG. 12O shows flow cytometry data for HEK 293FT reporter cells thathave the wild-type GUCY2D gene as the Cas9 target site and that are nottransfected with any DNA.

FIG. 12P shows flow cytometry data for HEK 293FT reporter cells thathave the R838H mutation as the Cas9 target site and that areco-transfected with (1) pSIA012 and (2) a Non-SIN-AAV SpCas9 (sEF1α).

FIG. 12Q shows flow cytometry data for HEK 293FT reporter cells thathave the R838H mutation as the Cas9 target site and that areco-transfected with (1) pSIA015 and (2) a Non-SIN-AAV SpCas9 (sEF1α).

FIG. 12R shows flow cytometry data for HEK 293FT reporter cells thathave the R838H mutation as the Cas9 target site and that are nottransfected with any DNA.

FIG. 12S shows flow cytometry data for HEK 293FT reporter cells thathave the R838C mutation as the Cas9 target site and that areco-transfected with (1) pSIA012 and (2) a Non-SIN-AAV SpCas9 (sEF1α).

FIG. 12T shows flow cytometry data for HEK 293FT reporter cells thathave the R838C mutation as the Cas9 target site and that areco-transfected with (1) pSIA015 and (2) Non-SIN-AAV SpCas9 (sEF1α).

FIG. 12U shows flow cytometry data for HEK 293FT reporter cells thathave the R838C mutation as the Cas9 target site and that are nottransfected with any sgRNA.

FIGS. 13A-C show western blot data for 3 different HEK 293FT reportercell lines that are co-transfected with either pSIA012, a plasmidcomprising an AAV sequence that encodes R838H_Sp_T2 sgRNA (a sgRNAcomprising SEQ ID NO: 5285) or pSIA015, a plasmid comprising an AAVsequence that encodes R838CH_Sp_T1 sgRNA (a sgRNA comprising SEQ ID NO:5398) and either (1) a SIN-AAV SpCas9 version 1 (sEF1α promoter), (2) aSIN-AAV SpCas9 version 2 (sEF1α promoter), or (3) a Non-SIN-AAV SpCas9(sEF1α promoter).

FIG. 13A is a western blot showing SpCas9, Actin, and GFP expression inHEK 293FT reporter cells that have the wild-type GUCY2D gene as the Cas9target site. These HEK 293FT reporter cells are transfected with eitherpSIA012 or pSIA015. The HEK 293FT reporter cells are also transfectedwith either (1) a SIN-AAV SpCas9 version 1 (sEF1α promoter), (2) aSIN-AAV SpCas9 version 2 (sEF1α promoter), or (3) a Non-SIN-AAV SpCas9(sEF1α promoter).

FIG. 13B is a western blot showing SpCas9, Actin, and GFP expression inHEK 293FT reporter cells that have the R838H mutation as the Cas9 targetsite. These HEK 293 FT reporter cells are transfected with eitherpSIA012 or pSIA015. The HEK 293FT reporter cells are also transfectedwith either (1) a SIN-AAV SpCas9 version 1 (sEF1α promoter), (2) aSIN-AAV SpCas9 version 2 (sEF1α promoter), or (3) a Non-SIN-AAV SpCas9(sEF1α promoter).

FIG. 13C is a western blot showing SpCas9, Actin, and GFP expression inHEK 293FT reporter cells that have the R838C mutation as the Cas9 targetsite. These HEK 293 FT reporter cells are transfected with eitherpSIA012 or pSIA015. The HEK 293FT reporter cells are also transfectedwith either (1) a SIN-AAV SpCas9 version 1 (sEF1α promoter), (2) aSIN-AAV SpCas9 version 2 (sEF1α promoter), or (3) a Non-SIN-AAV SpCas9(sEF1α promoter).

FIGS. 14A-C depict the structural arrangement of SIN-AAV SpCas9 version1 (GRK1 promoter), SIN-AAV SpCas9 version 2 (GRK1 promoter), andNon-SIN-AAV SpCas9 (GRK1 promoter).

FIG. 14A depicts the structural arrangement of SIN-AAV SpCas9 version 1(GRK1 promoter).

FIG. 14B depicts the structural arrangement of SIN-AAV SpCas9 version 2(GRK1 promoter).

FIG. 14C depicts the structural arrangement of Non-SIN-AAV SpCas9 (GRK1promoter).

FIGS. 15A-B are western blots showing SpCas9, Beta-Tubulin, and GFPexpression in cells isolated from mouse retinas that were previouslyinjected with either an AAV that encodes R838H_Sp_T2 sgRNA (a sgRNAcomprising SEQ ID NO: 5285) or AAV that encodes R838CH_Sp_T1 sgRNA (asgRNA comprising SEQ ID NO: 5398). Mouse retinas were co-injected withAAV-R838H (SEQ ID NO: 5481) and either (1) a SIN-AAV SpCas9 version 1(GRK1), (2) a SIN-AAV SpCas9 version 2 (GRK1), or (3) a Non-SIN-AAVSpCas9 (GRK1).

FIG. 15A is a western blot showing SpCas9, Beta-Tubulin, and GFPexpression in cells isolated from mouse retinas that were previouslyinjected with an AAV vector that encodes R838H_Sp_T2 sgRNA (a sgRNAcomprising SEQ ID NO: 5285); AAV-R838H (SEQ ID NO: 5481); and either (1)a SIN-AAV SpCas9 version 1 (GRK1), (2) a SIN-AAV SpCas9 version 2(GRK1), or (3) a Non-SIN-AAV SpCas9 (GRK1).

FIG. 15B is a western blot showing SpCas9, Beta-Tubulin, and GFPexpression in cells isolated from mouse retinas that were previouslyinjected with an AAV vector that encodes R838CH_Sp_T1 sgRNA (a sgRNAcomprising SEQ ID NO: 5398); AAV-R838H (SEQ ID NO: 5481); and either (1)a SIN-AAV SpCas9 version 1 (GRK1), (2) a SIN-AAV SpCas9 version 2(GRK1), or (3) a Non-SIN-AAV SpCas9 (GRK1).

FIG. 16 is a graph showing the editing efficiency for immortalized humanpatient-derived fibroblasts that have a R838H mutant allele as a copy ofthe GUCY2D gene, co-transfected with pSpCas9, a plasmid that encodesSpCas9, and either: pSIA012, pSIA015, or pSIA003.

FIG. 17 is a graph showing the editing efficiency for immortalized humanpatient-derived fibroblasts that have a R838C mutant allele as a copy ofthe GUCY2D gene, co-transfected with pSpCas9, a plasmid that encodesSpCas9, and either: pSIA012, pSIA015, or pSIA003.

FIG. 18 is a graph showing the editing efficiency for hTERT-immortalizedhuman fibroblast cells that are homozygous for the wild-type copy of theGUCY2D gene, co-transfected with pSpCas9, a plasmid that encodes SpCas9,and either: pSIA012, pSIA015, pSIA022, or pSIA003.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NOs: 1-612 are Cas endonuclease ortholog sequences.

SEQ ID NOs: 613-4696 are miRNA sequences.

SEQ ID NOs: 4697-5265 are AAV serotype sequences.

SEQ ID NO: 5266 is a GUCY2D nucleotide sequence.

SEQ ID NOs: 5267-5269 show sample sgRNA backbone sequences that SpCas9is complexed with.

SEQ ID NO: 5270 is a sample gRNA for a Streptococcus pyogenes Cas9endonuclease.

SEQ ID NO: 5271 shows a known family of homing endonuclease, asclassified by its structure.

SEQ ID NOs: 5272-5281 are 19-20 bp spacer sequences for targeting withinor near a GUCY2D gene or other DNA sequence that encodes a regulatoryelement of the GUCY2D gene with a S. pyogenes Cas9 endonuclease or S.aureus Cas9 endonuclease.

SEQ ID NOs: 5282-5293 are 19-20 bp spacer sequences for targeting withinor near a R838H mutation in a GUCY2D gene with a S. pyogenes Cas9endonuclease or S. aureus Cas9 endonuclease.

SEQ ID NOs: 5294-5303 are 19-20 bp spacer sequences for targeting withinor near a R838C mutation in a GUCY2D gene with a S. pyogenes Cas9endonuclease or S. aureus Cas9 endonuclease.

SEQ ID NOs: 5304-5313 are 19-20 bp spacer sequences for targeting withinor near a R838S mutation in a GUCY2D gene with a S. pyogenes Cas9endonuclease or S. aureus Cas9 endonuclease.

SEQ ID NOs: 5314-5355 are sequences that represent the target DNAsequences, for each of 42 sgRNA sequences in FIG. 2A.

SEQ ID NOs: 5356-5397 are sequences that represent the reverse strandsof the target DNA sequence to which the sgRNA will bind, for each of 42sgRNA sequences in FIG. 2A.

SEQ ID NOs: 5398-5409 are 19-20 bp spacer sequences for targeting withinor near a R838H mutation or R838C mutation in a GUCY2D gene with a S.pyogenes Cas9 endonuclease or Staphylococcus aureus Cas9 endonuclease.

SEQ ID NOs: 5410-5421 are sequences that represent the target DNAsequences, for each of 12 sgRNA sequences in FIG. 2D.

SEQ ID NOs: 5422-5433 are sequences that represent the reverse strandsof the target DNA sequence to which the sgRNA will bind, for each of 12sgRNA sequences in FIG. 2D.

SEQ ID NOs: 5434-5443 are 19-20 bp spacer sequences for targeting withinor near a R838H mutation or R838S mutation in a GUCY2D gene with a S.pyogenes Cas9 endonuclease or S. aureus Cas9 endonuclease.

SEQ ID NOs: 5444-5453 are sequences that represent the target DNAsequences, for each of 10 sgRNA sequences in FIG. 2D.

SEQ ID NOs: 5454-5463 are sequences that represent the reverse strandsof the target DNA sequence to which the sgRNA will bind, for each of 10sgRNA sequences in FIG. 2D.

SEQ ID NO: 5464 is a full-length sgRNA comprising SEQ ID NOs: 5285 and5267.

SEQ ID NO: 5465 is a full-length sgRNA comprising SEQ ID NOs: 5286 and5267.

SEQ ID NO: 5466 is a full-length sgRNA comprising SEQ ID NOs: 5398 and5267.

SEQ ID NO: 5467 does not include a sequence.

SEQ ID NO: 5468 is a plasmid comprising an AAV sequence that encodes fora sgRNA comprising SEQ ID NOs: 5274 and 5267.

SEQ ID NO: 5469 is pSIA012, a plasmid comprising an AAV sequence thatencodes for a sgRNA comprising SEQ ID NO: 5464.

SEQ ID NO: 5470 is a plasmid comprising an AAV sequence that encodes fora sgRNA comprising SEQ ID NO: 5465.

SEQ ID NO: 5471 is pSIA015, a plasmid comprising an AAV sequence thatencodes for a sgRNA comprising SEQ ID NO: 5466.

SEQ ID NO: 5472 is a plasmid sequence comprising SIN-AAV SpCas9 ver. 1(GRK1 promoter), depicted in FIG. 14A.

SEQ ID NO: 5473 is a plasmid sequence comprising SIN-AAV SpCas9 ver. 2(GRK1 promoter), depicted in FIG. 14B.

SEQ ID NO: 5474 is a plasmid sequence comprising Non-SIN-AAV SpCas9(GRK1 promoter), depicted in FIG. 14C.

SEQ ID NO: 5475 is a plasmid sequence comprising SIN-AAV SpCas9 ver. 1(sEF1α promoter), depicted in FIG. 11A.

SEQ ID NO: 5476 is a plasmid sequence comprising SIN-AAV SpCas9 ver. 2(sEF1α promoter), depicted in FIG. 11B.

SEQ ID NO: 5477 is a plasmid sequence comprising Non-SIN-AAV SpCas9(sEF1α promoter), depicted in FIG. 11C.

SEQ ID NO: 5478 is a possible SIN site located upstream of the SpCas9ORF in the SIN-AAV SpCas9 ver. 1, depicted in FIGS. 11A and 14A.

SEQ ID NO: 5479 is a possible SIN site located upstream of the SpCas9ORF in the SIN-AAV SpCas9 ver. 2, depicted in FIGS. 11B and 14B.

SEQ ID NO: 5480 is a possible SIN site located downstream of the SpCas9ORF in SIN-AAV SpCas9 ver. 1 depicted in FIGS. 11A and 14A anddownstream of the SpCas9 ORF in SIN-AAV SpCas9 ver. 2 depicted in FIGS.11B and 14B.

SEQ ID NO: 5481 is a possible SIN site located upstream of the SpCas9ORF in the SIN-AAV SpCas9 ver. 1, depicted in FIGS. 11A and 14A.

SEQ ID NO: 5482 is a possible SIN site located upstream of the SpCas9ORF in the SIN-AAV SpCas9 ver. 2, depicted in FIGS. 11B and 14B.

SEQ ID NO: 5483 is a possible SIN site located downstream of the SpCas9ORF in SIN-AAV SpCas9 ver. 1 depicted in FIGS. 11A and 14A anddownstream of the SpCas9 ORF in SIN-AAV SpCas9 ver. 2 depicted in FIGS.11B and 14B.

SEQ ID NO: 5484 is a possible SIN site located upstream of the SpCas9ORF in the SIN-AAV SpCas9 ver. 1, depicted in FIGS. 11A and 14A.

SEQ ID NO: 5485 is a possible SIN site located upstream of the SpCas9ORF in the SIN-AAV SpCas9 ver. 2, depicted in FIGS. 11B and 14B.

SEQ ID NO: 5486 is a possible SIN site located downstream of the SpCas9ORF in SIN-AAV SpCas9 ver. 1 depicted in FIGS. 11A and 14A anddownstream of the SpCas9 ORF in SIN-AAV SpCas9 ver. 2 depicted in FIGS.11B and 14B.

SEQ ID NO: 5487 is a possible SIN site located upstream of the SpCas9ORF in the SIN-AAV SpCas9 ver. 1, depicted in FIGS. 11A and 14A.

SEQ ID NO: 5488 is a possible SIN site located upstream of the SpCas9ORF in the SIN-AAV SpCas9 ver. 2, depicted in FIGS. 11B and 14B.

SEQ ID NO: 5489 is a possible SIN site located downstream of the SpCas9ORF in SIN-AAV SpCas9 ver. 1 depicted in FIGS. 11A and 14A anddownstream of the SpCas9 ORF in SIN-AAV SpCas9 ver. 2 depicted in FIGS.11B and 14B.

SEQ ID NO: 5490 is a possible SIN site located upstream of the SpCas9ORF in the SIN-AAV SpCas9 ver. 1, depicted in FIGS. 11A and 14A.

SEQ ID NO: 5491 is a possible SIN site located upstream of the SpCas9ORF in the SIN-AAV SpCas9 ver. 2, depicted in FIGS. 11B and 14B.

SEQ ID NO: 5492 is a possible SIN site located downstream of the SpCas9ORF in SIN-AAV SpCas9 ver. 1 depicted in FIGS. 11A and 14A anddownstream of the SpCas9 ORF in SIN-AAV SpCas9 ver. 2 depicted in FIGS.11B and 14B.

SEQ ID NO: 5493 is the sequence for AAV-R838H.

SEQ ID NOs: 5494-5505 show sample sgRNA backbone sequences that SaCas9is complexed with.

SEQ ID NO: 5506 is the AAV sequence in pSIA012.

SEQ ID NO: 5507 is the AAV sequence in pSIA015.

SEQ ID NO: 5508 is the AAV sequence in SIN-AAV-SpCas9 version 1 (GRK1).

SEQ ID NO: 5509 is the AAV sequence in SIN-AAV-SpCas9 version 2 (GRK1).

SEQ ID NO: 5510 is the AAV sequence in SIN-AAV-SpCas9 version 1 (sEF1α).

SEQ ID NO: 5511 is the AAV sequence in SIN-AAV-SpCas9 version 2 (sEF1α).

SEQ ID NO: 5512 is a plasmid sequence for pSpCas9 (BB)-2A-miRFP670(“pSpCas9”).

SEQ ID NO: 5513 is a spacer sequence for a gRNA or sgRNA encoded bypSIA003 from Examples 32-34.

SEQ ID NO: 5514 is pSIA022, a plasmid comprising an AAV sequence thatencodes for a sgRNA comprising SEQ ID NO: 5274.

DETAILED DESCRIPTION

Applicants have discovered a novel method for treating an autosomaldominant CORD, e.g., a CORD associated with a mutation in a GUCY2D gene.The method can result in slowing the development of CORD or preventingdevelopment of disease in an individual. Applicants have also discovereda self-inactivating CRISPR/Cas system.

Therapeutic Approach

The methods provided herein, regardless of whether a cellular, ex vivoor in vivo method can involve one or a combination of the followingmethods. One method involves reducing or eliminating expression of aGUCY2D allele containing a mutation (e.g., an R838H, R838C, or R838Smutation) at the protein level using non-homologous end joining (NHEJ)to introduce a frameshift mutation in the R838H, R838C, or R838S mutantallele. The frameshift can be caused by an insertion or deletion thatarises during NHEJ. In another method, a mutant allele (e.g., an R838H,R838C, or R838S mutation) is corrected by HDR. A third method includesknocking-in GUCY2D cDNA into a GUCY2D gene locus or into a safe harborlocus.

The NHEJ frameshifting strategy can involve inducing one single strandedbreak or double stranded break within or near the R838H, R838C, or R838Smutation in the GUCY2D gene with one or more CRISPR endonucleases and agRNA (e.g., crRNA+tracrRNA, or sgRNA), or two or more single strandedbreaks or double stranded breaks within or near the R838H, R838C, orR838S mutation in the GUCY2D gene with two or more CRISPR endonucleasesand two or more sgRNAs. This approach can prevent thetranscription/synthesis of the R838H, R838C, or R838S mutatant allele bycausing a frameshift in the R838H, R838C, or R838S mutant allele. Thismethod utilizes gRNAs or sgRNAs specific for the R838H, R838C, or R838Smutation in the GUCY2D gene.

The HDR correction strategy can involve inducing one single strandedbreak or double stranded break within or near the R838H, R838C, or R838Smutation in the GUCY2D gene with one or more CRISPR endonucleases and agRNA (e.g., crRNA+tracrRNA, or sgRNA), or two or more single strandedbreaks or double stranded breaks within or near the R838H, R838C, orR838S mutation in the GUCY2D gene with one or more CRISPR endonucleases(Cas9, Cpf1 and the like) and two or more gRNAs, in the presence of adonor DNA template introduced exogenously to direct the cellular DSBresponse to Homology-Directed Repair. The donor DNA template can be ashort single stranded oligonucleotide, a short double strandedoligonucleotide, a long single or double stranded DNA molecule. Themethods can provide gRNA pairs that make a deletion by cutting the genetwice, one gRNA cutting at the 5′ end of the R838H, R838C, or R838Smutation and the other gRNA cutting at the 3′ end of the R838H, R838C,or R838S mutation that facilitates insertion of a new sequence from apolynucleotide donor template to replace the R838H, R838C, or R838Smutation in the GUCY2D gene. The cutting can be accomplished by a pairof DNA endonucleases that each makes a DSB (one DSB on each end of theR838H, R838C, or R838S mutation), or by multiple nickases that togethermake a DSB (one DSB on each end of the R838H, R838C, or R838S mutation).This method utilizes gRNAs or sgRNAs specific for the R838H, R838C, orR838S mutation in the GUCY2D gene and donor DNA molecules.

The knock-in strategy involves knocking-in GUCY2D cDNA into the GUCY2Dgene locus using a gRNA (e.g., crRNA+tracrRNA, or sgRNA) or a pair ofgRNAs targeting upstream of or in the first or other exon and/or intronof the GUCY2D gene, or in a safe harbor site (such as AAVS1). The donorDNA can be single or double stranded DNA.

The advantages for the above strategies (frameshift, correction, andknock-in) are similar, including in principle both short and long termbeneficial clinical and laboratory effects. The knock-in approachprovides at least one advantage over the frameshift and correctionapproach—the ability to treat all patients versus only a subset ofpatients.

Such methods use endonucleases, such as CRISPR-associated (Cas9, Cpf1and the like) nucleases, to stably correct the R838H, R838C, or R838Smutation within the genomic locus of the GUCY2D gene. Any CRISPRendonuclease can be used in the methods of the present disclosure, eachCRISPR endonuclease having its own associated PAM, which can or cannotbe disease specific. For example, gRNA spacer sequences for targetingthe R838H, R838C, or R838S mutation in the GUCY2D gene with aCRISPR/Cas9 endonuclease from S. pyogenes or S. aureus have beenidentified in SEQ ID NOs. 5282-5313, 5398-5409, and 5434-5443 of theSequence Listing.

Examples set forth in the present disclosure can induce single strandedbreaks or double stranded breaks within or near the R838H, R838C, orR838S mutation in the GUCY2D gene to introduce a frameshift or correctthe R838H, R838C, or R838S mutation within the GUCY2D gene with as fewas a single treatment (rather than deliver potential therapies for thelifetime of the patient).

Cone-Rod Dystrophy (CORD)

Cone rod dystrophies (CORD) are a group of inherited retinal dystrophiescharacterized by retinal pigment deposits predominantly localized in themacular region. CORD leads to primary degeneration of cones followed byloss of rods and has a prevalence of 1:40,000 (Garcia-Hoyos et al.,2011, Molecular Vision; Mukherjee et al., 2014, Eye, 28:481-487). Someforms of CORD have been observed to be inherited as an autosomaldominant, autosomal recessive or X-linked recessive trait while othersoccur spontaneously. Onset of the disease normally occurs in the firsttwo decades of life. There are four major causative genes involved inCORD: ABCA4 (causing 30-60% of all autosomal recessive CORDs), CRX andGUCY2D (responsible for 35% of autosomal dominant CORDs/conedystrophies), and RPGR (causing X-linked CORDs). All GUCY2D genemutations in patients with autosomal dominant CORD are located at codon838 or the two adjacent codons 837 and 839, leading to degeneration ofphotoreceptors and visual deterioration (Mukherjee et al., 2014, Eye,28:481-487; Kitiratschky et al., 2008, Science, 49: 5015-5023).Mutations in the GUCY2D gene account for 20% of all CORD disease.Mutations in codon 838 account for up to 95% of all mutations in theGUCY2D gene leading to photoreceptor degeneration due to the mutatedRetCG-1 protein. In the US itself there are 1,500 patients with codon838 mutations in the GUCY2D gene (Garcia-Hoyos et al., 2011, MolecularVision 17: 1103-1109; Mukherjee et al., 2014, Eye, 28:481-487;Kitiratschky et al., 2008, Science, 49: 5015-5023), with 19 new patientsper year (Hamel et al., 2006, Orphanet Journal of Rare Diseases 2: 1-7).

A hallmark of CORDs is retinal pigment deposits visible on fundusexamination, predominantly localized in the macular region (Hamel etal., 2006, Orphanet Journal of Rare Diseases 2: 1-7). Most CORD patientsdisplay the following symptoms: decreased central vision, color visiondefects, photophobia and decreased sensitivity in the central field atearly stages, followed by progressive loss in peripheral vision andnight blindness at later stages. The clinical course of CORDs isgenerally more severe and rapid than of rod cone dystrophies, leading toearlier legal blindness and disability (Hamel et al., 2006, OrphanetJournal of Rare Diseases 2: 1-7; Mukherjee et al., 2014, Eye,28:481-487). Non syndromic CORDs are genetically heterogenous and may beinherited as autosomal dominant, autosomal recessive, or X-linked trait(Mukherjee et al., 2014, Eye, 28:481-487).

CORD can cause a variety of symptoms that include decreased centralvisual acuity and photophobia which is a reduced ability to see colorsand an increased sensitivity to light, both of which are the firstsymptoms to appear in the first decasde of life, followed by nightblindness and further decrease of visual acuity. CORD can be dividedinto two groups, stationary CORD in which symptoms remain stable and aremostly present at birth or early childhood, and progressive CORD inwhich symptoms slowly become worse over time, so that a person isconsidered legally blind with a visual acuity that is 20/200 or worse.Progression of CORD is generally more severe and rapid than rod conedystrophy (e.g. retinitis pigmentosa), leading to earlier blindness.Total blindness is not common in individuals affected with conedystrophy and peripheral vision is usually unaffected. Affectedindividuals can see well at night or under conditions of low light asthe rod cells are usually unaffected. Key endpoints to assess treatmentrelated outcomes include measuring visual acuity, electroretinographyresponse, optical coherence tomography and patient reported outcome.

CORD caused by a R838H, R838C, or R838S mutation in a GUCY2D gene is amonogenic disorder with autosomal dominant inheritance. If a patientonly has one R838H, R838C, or R838S mutant allele, a frameshift can beintroduced into one R838H, R838C, or R838S mutant allele per cell toprevent the transcription/synthesis of the one R838H, R838C, or R838Smutant allele. A novel approach has been discovered for ameliorating theeffects of autosomal dominant CORD by introducing a frameshift into oneR838H, R838C, or R838S mutant allele per cell to prevent thetranscription/synthesis of the one R838H, R838C, or R838S mutant allele.

Also, if a patient only has one R838H, R838C, or R838S mutant allele,the one R838H, R838C, or R838S mutant allele can be corrected to restoreRetGC1 function. If a patient has two R838H, R838C, or R838S mutantalleles, both R838H, R838C, or R838S mutant alleles can be correctedwith HDR to restore RetGC1 function.

Introducing a frameshift into a R838H, R838C, or R838S mutant allele orcorrecting a R838H, R838C, or R838S mutant allele using gene editingprovides an important improvement over existing or potential therapies,such as introduction of RetGC1 expression cassettes through lentivirusdelivery and integration because of its preciseness and lower adverseeffects.

Guanylate Cyclase 2D (GUCY2D) Gene

The GUCY2D gene spans a sequence of approximately 17.7 kb, ranges from8,002,594-8,020,339 bp, and encodes for the protein retinal membraneguanylate cyclase-1 (RetGC1) that functions in the maintenance of normalvision and is located within the photoreceptors (expressed in both rodand cone photoreceptors) in the retina. The protein is predominantlyexpressed in cone outer segments and has a molecular weight of 120 Kd.The GUCY2D cDNA consists of 3641 nucleotides, spanning 17.7 kb ofgenomic DNA. RetGC-1 takes part in the recovery phase ofphototransduction and is located in the marginal region of the cone'souter segments. RetGC proteins play an important role in restoringphotoreceptor sensitivity due to their involvement in the synthesis ofcGMP, and regulate the calcium level in cells. Once light enters theeye, it stimulates specialized pigments in photoreceptor cells whichfurther trigger a series of chemical reactions that produce anelectrical signal. This electrical signal is decoded by the brain asvision. Following stimulation, the photoreceptors return to theirresting state and await the next round of stimulation. RetGC-1 proteintakes part in a chemical reaction that helps return photoreceptors totheir resting state after light exposure (Boye, 2016, Advances inExperimental Medicine and Biology). Light stimulates the degradation ofcGMP, causing the closing of cation channels, which results in cellhyperpolarization and neurotransmitter release. At lower concentration,calcium stimulates RetGCs and as a consequence the cGMP level isrestored. As a result, the cation channels reopen and photosensitivityis restored to the cell (Garcia-Hoyos et al., 2011, Molecular Vision 17:1103-1109).

So far, several identified GUCY2D mutations in CORD patients are locatedat the codon 838 or the two adjacent ones 837, and 839 (Mukherjee etal., 2014, Eye, 28:481-487; Garcia-Hoyos et al., 2011, Molecular Vision17: 1103-1109). R838X mutations and adjacent mutations induce a gain offunction of RetGC-1 leading to higher calcium concentration and inducingphotoreceptor degeneration (Garcia-Hoyos et al., 2011, Molecular Vision17: 1103-1109; Mukherjee et al., 2014, Eye, 28:481-487; Hamel et al.,2007, Orphanet Journal of Rare Diseases). Until now, several identifiedGUCY2D mutations in CORD patients are located at codon 838 or the twoadjacent codons 837 and 839 (Mukherjee et al., 2014, Eye, 28:481-487;Garcia-Hoyos et al., 2011, Molecular Vision 17: 1103-1109) leading todegeneration of photoreceptors and visual deterioration. These mutationsproduce a gain of function in increasing the affinity of the RetGC-1 forthe guanylate cyclase activating proteins even in high calciumconcentration, leading to a higher cGMP concentration (Mukherjee et al.,2014, Eye, 28:481-487; Garcia-Hoyos et al., 2011, Molecular Vision 17:1103-1109; Weigell-Weber et al., 2000, Archives of Ophthalmology; VanGhelue et al., 2000, Ophthalmic Genetics; Wilkie et al., 2000, HumanMolecular Genetics). The photoreceptor death is believed to be caused bythe high cGMP concentration keeping cGMP-gated cation channel open,resulting in an increased calcium concentration in the cell (Mukherjeeet al., 2014, Eye, 28:481-487; Tucker et al., 1999, Proceedings of theNational Academy of Sciences, 96:9039-9044).

GUCY2D can also be referred to as GUC1A4; RCD2; LCA1; GUC2D; retGC; LCA;ROSGC; CYGD; RETGC-1; CORD6; ROS-GC1; RETGC; CORD5; RETGC1; GuanylateCyclase 2D, Membrane (Retina-Specific); Rod Outer Segment MembraneGuanylate Cyclase; Guanylate Cyclase 2D, Retinal; Retinal GuanylateCyclase 1; Cone Rod Dystrophy 6; EC 4.6.1.2; RETGC-1; GUC1A4; ROS-GC;RetGC; CORD6; GUC2D; EC 4.6.1; ROS-GC1; RETGC1; CORD5; ROSGC; CYGD;LCA1; RCD2; LCA. GUCY2D has a cytogenetic location of 17p13.1 and thegenomic coordinates as seen on Ensembl database are on Chromosome 17 onthe forward strand at position 8,002,594-8,020,339. A nucleotidesequence of GUCY2D is shown as SEQ ID NO: 5266. RP11-1099M24.8 is thegene upstream of GUCY2D on the forward strand and ALOX15B is the genedownstream of GUCY2D on the reverse strand. Contained within the GUCY2Dgene is the gene RP11-474L23.3 on the reverse strand. The geneRP11-1099M24.9 is located on the reverse strand opposite of GUCY2D.GUCY2D has a NCBI gene ID of 3000, Uniprot ID of Q02846 and Ensembl GeneID of ENSG00000132518. GUCY2D has 1,684 SNPs, 19 introns and 20 exons.The exon identifier from Ensembl and the start/stop sites of the intronsand exons are shown in Table 1.

TABLE 1 Exons and Introns for GUCY2D Exon Start/Stop No. Exon ID No.Intron Intron based on Exon ID Start/Stop EX1 ENSE00001365187 8,002,594-INT1 Intron ENSE00001365187- 8,002,735- 8,002,734 ENSE000013008118,003,038 EX2 ENSE00000905467 8,003,852- INT2 Intron ENSE00001300811-8,003,769- 8,004,156 ENSE00000905467 8,003,851 EX3 ENSE000009054808,015,743- INT3 Intron ENSE00000905467- 8,004,157- 8,015,841ENSE00001308194 8,006,362 EX4 ENSE00001293688 8,007,931- INT4 IntronENSE00001308194- 8,006,715- 8,008,032 ENSE00001330137 8,007,059 EX5ENSE00001293922 8,014,859- INT5 Intron ENSE00001330137- 8,007,145-8,015,051 ENSE00001305434 8,007,425 EX6 ENSE00001300099 8,012,144- INT6Intron ENSE00001305434- 8,007,529- 8,012,350 ENSE00001293688 8,007,930EX7 ENSE00001300811 8,003,039- INT7 Intron ENSE00001293688- 8,008,033-8,003,768 ENSE00001311972 8,009,505 EX8 ENSE00001305434 8,007,426- INT8Intron ENSE00001311972- 8,009,587- 8,007,528 ENSE00001300099 8,012,143EX9 ENSE00001306526 8,013,103- INT9 Intron ENSE00001300099- 8,012,351-8,013,252 ENSE00001321424 8,012,449 EX10 ENSE00001308194 8,006,363-INT10 Intron ENSE00001321424- 8,012,607- 8,006,714 ENSE000013065268,013,102 EX11 ENSE00001310706 8,015,328- INT11 Intron ENSE00001306526-8,013,253- 8,015,502 ENSE00001328414 8,013,879 EX12 ENSE000013113878,014,601- INT12 Intron ENSE00001328414- 8,014,029- 8,014,764ENSE00001311387 8,014,600 EX13 ENSE00001311972 8,009,506- INT13 IntronENSE00001311387- 8,014,765- 8,009,586 ENSE00001293922 8,014,858 EX14ENSE00001312940 8,016,205- INT14 Intron ENSE00001293922- 8,015,052-8,016,290 ENSE00001310706 8,015,327 EX15 ENSE00001317612 8,016,443-INT15 Intron ENSE00001310706- 8,015,503- 8,016,554 ENSE000009054808,015,742 EX16 ENSE00001321424 8,012,450- INT16 Intron ENSE00000905480-8,015,842- 8,012,606 ENSE00001329972 8,015,926 EX17 EN5E000013284148,013,880- INT17 Intron EN5E00001329972- 8,016,022- 8,014,028ENSE00001312940 8,016,204 EX18 EN5E00001329972 8,015,927- INT18 IntronENSE00001312940- 8,016,291- 8,016,021 ENSE00001317612 8,016,442 EX19EN5E00001330137 8,007,060- INT19 Intron ENSE00001317612- 8,016,555-8,007,144 ENSE00001390086 8,020,127 EX20 EN5E00001390086 8,020,128-8,020,339

Table 2 provides information on all of the transcripts for the GUCY2Dgene based on the Ensembl database. Provided in Table 2 are thetranscript ID from Ensembl and corresponding NCBI RefSeq ID for thetranscript, the translation ID from Ensembl and the corresponding NCBIRefSeq ID for the protein, the biotype of the transcript sequence asclassified by Ensembl and the exons and introns in the transcript basedon the information in Table 1.

TABLE 2 Transcript Information for GUCY2D Transcript Protein NCBI NCBIExon ID Intron ID Transcript RefSeq Translation RefSeq Sequence fromfrom ID ID ID ID Biotype Table 1 Table 1 ENST0000 NM_000180 ENSP00000NP_000171 Protein EX1, EX2, EX3, INT1, INT2, INT3, 0254854.4 254854coding EX4, EX5, EX6, INT4, INT5, INT6, EX7, EX8, EX9, INT7, INT8, INT9,EX10, EX11, INT10, INT11, EX12, EX13, INT12, INT13, EX14, EX15, INT14,INT15, EX16, EX17, INT16, INT17, EX18, EX19, INT18, INT19 EX20 ENST0000— — Retained EX20, EX21 INT20 0574510.1 intron

GUCY2D has 1,684 SNPs and the NCBI rs number and/or UniProt VAR numberfor the SNPs of the GUCY2D gene are rs2534, rs2816, rs3813585,rs3829789, rs3891083, rs3928731, rs4791452, rs4791456, rs4792111,rs7406106, rs7501868, rs7503918, rs8068722, rs8069344, rs8071166,rs9889612, rs9891137, rs9891219, rs9905393, rs9905402, rs9912176,rs9914315, rs9914686, rs9914937, rs11655487, rs11655691, rs12103471,rs12103519, rs12103521, rs12449814, rs12602083, rs28743021, rs28933695,rs33914314, rs33942683, rs34016036, rs34049818, rs34065746, rs34331388,rs34463160, rs34466558, rs34594470, rs34596269, rs34598902, rs34671919,rs34922798, rs35134646, rs35146471, rs35357335, rs35616384, rs35881051,rs35883105, rs55687426, rs55916957, rs56034424, rs56056557, rs56130505,rs56280231, rs56316238, rs56348143, rs57184071, rs57273310, rs58765829,rs60130989, rs61749663, rs61749664, rs61749665, rs61749667, rs61749668,rs61749669, rs61749670, rs61749671, rs61749672, rs61749673, rs61749674,rs61749675, rs61749676, rs61749677, rs61749678, rs61749679, rs61749680,rs61749681, rs61749682, rs61749683, rs61749753, rs61749754, rs61749755,rs61749756, rs61749758, rs61749759, rs61750160, rs61750161, rs61750162,rs61750163, rs61750164, rs61750166, rs61750167, rs61750168, rs61750169,rs61750170, rs61750171, rs61750172, rs61750173, rs61750174, rs61750175,rs61750176, rs61750177, rs61750178, rs61750179, rs61750180, rs61750181,rs61750182, rs61750183, rs61750184, rs61750185, rs61750186, rs61750187,rs61750188, rs61750189, rs61750190, rs61750192, rs61750193, rs61750194,rs61750196, rs61750197, rs61750198, rs61750199, rs62065069, rs62065070,rs62641254, rs63340060, rs63749076, rs63749078, rs67594392, rs72203439,rs72841478, rs72841480, rs72841481, rs72841482, rs73237655, rs73244190,rs73978651, rs73978652, rs73978653, rs73978654, rs73978655, rs73978656,rs73978658, rs73978659, rs74579703, rs74656480, rs74864625, rs75290069,rs75837616, rs76196120, rs76459165, rs77430877, rs77752392, rs77922915,rs78117741, rs78380494, rs78434534, rs78761797, rs78844078, rs78901930,rs78908751, rs79288861, rs79347759, rs79581544, rs79887212, rs79980119,rs80076597, rs80245692, rs112085163, rs112360110, rs112372281,rs112764660, rs112923697, rs112984002, rs113031167, rs113153323,rs113332317, rs115598390, rs116236245, rs116728094, rs116841812,rs116870332, rs117241209, rs117853745, rs118026892, rs118057940,rs118102619, rs118140564, rs137853897, rs138162268, rs138176835,rs138200238, rs138255027, rs138298187, rs138596240, rs138635198,rs138836357, rs138869083, rs138922415, rs139019420, rs139046650,rs139168077, rs139616184, rs139731548, rs139763939, rs140005435,rs140366544, rs140436048, rs140628227, rs140638938, rs140657975,rs140661218, rs140889612, rs140936694, rs140991876, rs141214199,rs141229863, rs141346556, rs141352623, rs141360883, rs141592651,rs141837808, rs141917297, rs141956583, rs141967896, rs142050758,rs142207894, rs142275378, rs142351773, rs142368822, rs142415521,rs142997995, rs143323176, rs143524082, rs143535791, rs143585840,rs143604121, rs143607596, rs143650826, rs143730352, rs143745703,rs143761257, rs143938678, rs144151076, rs144291605, rs144349779,rs144442115, rs144458688, rs144565168, rs144659131, rs145060888,rs145344081, rs145420245, rs145717676, rs146031822, rs146149224,rs146406238, rs146570135, rs146820642, rs146849545, rs146855363,rs146872553, rs147017233, rs147164228, rs147166962, rs147201985,rs147410617, rs147586061, rs147656459, rs148136213, rs148394581,rs148448937, rs148624438, rs148761225, rs148871664, rs148924873,rs148987106, rs149149530, rs149260011, rs149314785, rs149560134,rs149722832, rs149866657, rs150173998, rs150185423, rs150422660,rs150742659, rs150797198, rs151052136, rs151079263, rs151106252,rs151126238, rs151268449, rs151330485, rs180740641, rs180796231,rs180896227, rs181356213, rs181566410, rs181567056, rs181800610,rs182105834, rs182237936, rs182456792, rs183308730, rs183331808,rs183393377, rs183480892, rs183556945, rs183561005, rs184130107,rs184151004, rs184164679, rs184234997, rs184286345, rs184725910,rs184811496, rs185088930, rs185157201, rs185513239, rs185569607,rs185713115, rs185920216, rs186268358, rs186335397, rs186508466,rs186802043, rs186826861, rs186938529, rs187098034, rs187499915,rs187668100, rs187761992, rs187833219, rs187834138, rs187999872,rs188568530, rs188638994, rs188708948, rs188779114, rs189065870,rs189141326, rs189183021, rs189559705, rs189605015, rs189807747,rs189930296, rs189984380, rs190283426, rs190420345, rs190468215,rs190680904, rs190700778, rs190851646, rs191165076, rs191204702,rs191332697, rs191576243, rs191818936, rs191907268, rs192007830,rs192059023, rs192306625, rs192765108, rs192836968, rs192859514,rs193175998, rs193212053, rs199611541, rs199708312, rs199817768,rs199828903, rs199835050, rs199912675, rs199931193, rs199953653,rs199966203, rs200032594, rs200128473, rs200189360, rs200211315,rs200215575, rs200241218, rs200403362, rs200558780, rs200586401,rs200637525, rs200651999, rs200700723, rs200705417, rs200855025,rs200886585, rs201008187, rs201054971, rs201090802, rs201119605,rs201196538, rs201294458, rs201319533, rs201383207, rs201388569,rs201414567, rs201541863, rs201587670, rs201656108, rs201717870,rs201897109, rs202094105, rs202111469, rs202132636, rs267606857,rs281865408, rs281865409, rs281865410, rs281865411, rs281865412,rs281865413, rs367660008, rs367711120, rs367755752, rs367767119,rs367817490, rs368301973, rs368330301, rs368349186, rs368384232,rs368480652, rs368557900, rs368799458, rs368916122, rs368923721,rs368944056, rs368945604, rs368958527, rs369035095, rs369222553,rs369247789, rs369315814, rs369527655, rs369547545, rs369607137,rs369663256, rs369920240, rs370054772, rs370166526, rs370291650,rs370295773, rs370303747, rs370306654, rs370318503, rs370350737,rs370607318, rs370725838, rs370742162, rs370833664, rs370916719,rs371160436, rs371176908, rs371322812, rs371367958, rs371458632,rs371533971, rs371541944, rs371565742, rs371677282, rs371718267,rs371908939, rs371919912, rs371952237, rs372005126, rs372011559,rs372093845, rs372118691, rs372151247, rs372189031, rs374354168,rs374464654, rs375010731, rs375105072, rs375259185, rs375319109,rs375424336, rs375428889, rs375468242, rs375574116, rs375686386,rs375727197, rs375760706, rs375851554, rs376006766, rs376062280,rs376076112, rs376337508, rs376364350, rs376439753, rs376468724,rs376479795, rs376508683, rs376601845, rs376615794, rs376759049,rs376861829, rs376929203, rs376992406, rs377021127, rs377103968,rs377199837, rs377279983, rs377287956, rs374969713, rs374927150,rs374924527, rs374800328, rs374679518, rs374658427, rs374636320,rs374580575, rs374566845, rs374520619, rs374515716, rs374507808,rs374507426, rs374505063, rs374138416, rs374065209, rs374031991,rs373866646, rs373827556, rs373788984, rs373767265, rs373615436,rs373533477, rs373468027, rs373400629, rs373362030, rs373357165,rs373010281, rs372902332, rs372651614, rs372631047, rs372543612,rs372437131, rs377299382, rs377349549, rs377388031, rs377557911,rs377568220, rs377594823, rs377617525, rs377622295, rs377648185,rs377650196, rs386834239, rs397700887, rs398123233, rs527329237,rs527542013, rs527646004, rs527659830, rs527767509, rs528169554,rs528203345, rs528258925, rs528791875, rs528826742, rs529037612,rs529180514, rs529232821, rs529348303, rs529594203, rs529615133,rs529702465, rs529726426, rs529993769, rs530057359, rs530185073,rs530209613, rs530328021, rs531023711, rs531091295, rs531217355,rs531378185, rs531556241, rs531612488, rs531669033, rs531693520,rs531982313, rs532142507, rs532153534, rs532339805, rs532388657,rs532466020, rs532577225, rs532826586, rs533083983, rs533311236,rs533407112, rs533940473, rs534022358, rs534195509, rs534270598,rs534452349, rs534482693, rs534505519, rs534591759, rs535329733,rs535365241, rs535373419, rs535800696, rs535966503, rs535982563,rs536011574, rs536530182, rs536731863, rs536792434, rs536803598,rs536805410, rs537127961, rs537485807, rs537729498, rs537757201,rs537797103, rs537928701, rs537953527, rs537992694, rs538000618,rs538161935, rs538470494, rs538719629, rs538856385, rs539014522,rs539110487, rs539380013, rs539468342, rs539558334, rs539723740,rs539797071, rs539967957, rs540118139, rs540262740, rs540414225,rs540448686, rs540833632, rs540877234, rs540877830, rs540911894,rs540943749, rs541055967, rs541073541, rs541299023, rs541449650,rs541537384, rs541807865, rs541841155, rs541897718, rs542322730,rs542436190, rs542466976, rs542520858, rs542570988, rs542720789,rs542922281, rs543329741, rs543370993, rs543456744, rs543802677,rs543919081, rs543940794, rs544071266, rs544365402, rs544368608,rs544410518, rs544410836, rs544448494, rs544905777, rs544938122,rs545104656, rs545271894, rs545349538, rs545438074, rs545438123,rs546016309, rs546260863, rs546323421, rs546376402, rs547778415,rs547846849, rs547867709, rs547885527, rs547968529, rs548079477,rs548161414, rs548396303, rs548756390, rs548929399, rs548945470,rs549100277, rs549142397, rs549834851, rs549995044, rs550028183,rs550073930, rs550107422, rs550857471, rs550915196, rs550992644,rs550996386, rs551078742, rs551592000, rs551713029, rs551724597,rs551798084, rs552064079, rs552184470, rs552697544, rs552738572,rs552850691, rs547441462, rs547112247, rs546983953, rs546875049,rs546466375, rs546417010, rs546378331, rs552923753, rs553072849,rs553310926, rs553602736, rs553760068, rs553793163, rs553866555,rs553908583, rs554046976, rs554078433, rs554409602, rs554451844,rs554782032, rs555035210, rs555042960, rs555455408, rs555571688,rs555835721, rs555903665, rs555980737, rs556070337, rs556242786,rs556306222, rs556306503, rs556475744, rs556748323, rs557073963,rs557108466, rs557241953, rs557387508, rs557434200, rs557461086,rs557610631, rs558084054, rs558182614, rs558202780, rs558231853,rs558292964, rs558294899, rs558330360, rs558513244, rs558953933,rs559102087, rs559483603, rs559542629, rs559924290, rs560225721,rs560270873, rs560366032, rs560379930, rs560407738, rs560476139,rs560548493, rs561318435, rs561600004, rs561715780, rs562248093,rs562265516, rs562446256, rs562578337, rs562580870, rs562685254,rs562730568, rs562794616, rs562931210, rs562955779, rs563005151,rs563014439, rs563091520, rs563093147, rs563208176, rs563600965,rs563639504, rs563773522, rs563790636, rs563853837, rs563889760,rs564138931, rs564461174, rs564535411, rs564575395, rs564644984,rs564809875, rs564839085, rs564868510, rs564922369, rs565045308,rs565490655, rs565683921, rs565948960, rs566271985, rs566465559,rs566728183, rs566769575, rs566891487, rs566928694, rs566944947,rs566953664, rs566986521, rs567294414, rs567308384, rs567402015,rs567463643, rs567651225, rs567708710, rs568061033, rs568220391,rs568227769, rs568241298, rs568389697, rs568725951, rs569133278,rs569288698, rs569335925, rs569363032, rs569378426, rs569402782,rs569613224, rs569618690, rs570251619, rs570326099, rs570329813,rs570604735, rs570648500, rs571077663, rs571108752, rs571236000,rs571359815, rs571420864, rs571522042, rs571876720, rs571940488,rs572166519, rs572214888, rs572363136, rs572527369, rs572687257,rs573270795, rs573367793, rs573405328, rs573455326, rs573465617,rs573715107, rs573883571, rs574106259, rs574248181, rs574350498,rs574862256, rs574872059, rs574894754, rs574926836, rs575137886,rs575674171, rs575870524, rs576181574, rs576275667, rs576719987,rs576720162, rs577011007, rs577023175, rs577407924, rs577720921,rs577797978, rs577800522, rs577806343, rs577880164, rs577906096,rs577918795, rs578216431, rs745306850, rs745306861, rs745419505,rs745426085, rs745444949, rs745511852, rs745533728, rs745551727,rs745618018, rs745627220, rs745710183, rs745761477, rs745816219,rs745816355, rs745882425, rs745890471, rs745897683, rs745956207,rs745956774, rs745965625, rs746002871, rs746020263, rs746058533,rs746068022, rs746150760, rs746244895, rs746245709, rs746326156,rs746361123, rs746387733, rs746463069, rs746477554, rs746525316,rs746631257, rs746671374, rs746733885, rs746760406, rs746769693,rs746806801, rs746827007, rs746859702, rs746883969, rs746893543,rs746924059, rs746947825, rs747008057, rs747038879, rs747095961,rs747098258, rs747148534, rs747173302, rs747300481, rs747338144,rs747342259, rs747354016, rs747387196, rs747391169, rs747534233,rs747611318, rs747646491, rs747660548, rs747728750, rs747733556,rs747807494, rs747809048, rs747899948, rs747948522, rs747951577,rs747953612, rs748013570, rs748043131, rs748156659, rs748202022,rs748255432, rs748301943, rs748509676, rs748520738, rs748564718,rs748588464, rs748653489, rs748665297, rs748677206, rs748789502,rs748798324, rs748817525, rs748820854, rs748946901, rs749012764,rs749084260, rs749101279, rs749138289, rs749164572, rs749240455,rs749314383, rs749432012, rs749435918, rs749436007, rs749463421,rs749511762, rs749535904, rs749607771, rs749621660, rs749651871,rs749736899, rs749800225, rs749863195, rs749912018, rs749966010,rs749991246, rs749999803, rs750005559, rs750007435, rs750153057,rs750247123, rs750301530, rs750336532, rs750399742, rs750399947,rs750453350, rs750454050, rs750484873, rs750566089, rs750572766,rs750635086, rs750668023, rs750743574, rs750889782, rs750895890,rs750904732, rs750906839, rs750967765, rs751090137, rs751132098,rs751263915, rs751295073, rs751340355, rs751389375, rs751520851,rs751582497, rs751705225, rs751794453, rs751802666, rs751811525,rs751822337, rs751882664, rs751992560, rs751995887, rs752037112,rs752057528, rs752088799, rs752141508, rs752146300, rs752269926,rs752327486, rs752420414, rs752521534, rs752607737, rs752627946,rs752715015, rs752736704, rs752751984, rs752758326, rs752784361,rs752804462, rs752812981, rs752820485, rs752935089, rs752946790,rs752996693, rs753057356, rs753164211, rs753166398, rs753166496,rs753167035, rs753220125, rs753282322, rs753291515, rs753328828,rs753445019, rs753447830, rs753468278, rs753488664, rs753507183,rs753600757, rs753620111, rs753660903, rs753750417, rs753766842,rs753855098, rs753886745, rs753928228, rs754025464, rs754134176,rs754170292, rs754170656, rs754193560, rs754266653, rs754274583,rs754320374, rs754329901, rs754431996, rs754473375, rs754581545,rs754709344, rs754717527, rs754747609, rs754800041, rs754833828,rs754841103, rs754869603, rs754954638, rs754964975, rs754978631,rs755056200, rs755071207, rs755072380, rs755088085, rs755150485,rs755184357, rs755223158, rs755324667, rs755329222, rs755414746,rs755454531, rs755464893, rs755515660, rs755519877, rs755548192,rs755696177, rs755696904, rs755768308, rs755787827, rs755908164,rs755999834, rs756031060, rs756031378, rs756042293, rs756042481,rs756044745, rs756121043, rs756123157, rs756135232, rs756210907,rs756319569, rs756393266, rs756464199, rs756464730, rs756478965,rs756575304, rs756639026, rs756677163, rs756693094, rs756695371,rs756730335, rs756733337, rs756787049, rs756851759, rs757041611,rs757104555, rs757158931, rs757230483, rs757250994, rs757251193,rs757257247, rs757259115, rs757273041, rs757310094, rs757387072,rs757466277, rs757508954, rs757525165, rs757589496, rs757657345,rs757720386, rs757724509, rs757823463, rs757835419, rs757871358,rs757884734, rs757925587, rs757948866, rs757949564, rs758113854,rs758260297, rs758326916, rs758442320, rs758522855, rs758602959,rs758629545, rs758645364, rs758659507, rs758775976, rs758841128,rs758931689, rs758939310, rs758941038, rs758982450, rs759011004,rs759075619, rs759135596, rs759158538, rs759173115, rs759220063,rs759253167, rs759280279, rs759370090, rs759501515, rs759511711,rs759516562, rs759544156, rs759562174, rs759564156, rs759619180,rs759723474, rs759731211, rs759777872, rs759778209, rs759948249,rs759949982, rs759951639, rs759957436, rs760016257, rs760105515,rs760107054, rs760126408, rs760126540, rs760126924, rs760179252,rs760202269, rs760285694, rs760307098, rs760397638, rs760426420,rs760472338, rs760494327, rs760501583, rs760521771, rs760662744,rs760681687, rs760765585, rs760766230, rs760877146, rs760937197,rs760943859, rs761039951, rs761153188, rs761200249, rs761213112,rs761309365, rs761408010, rs761445993, rs761448586, rs761484472,rs761498977, rs761513763, rs761529451, rs761542444, rs761543634,rs761631031, rs761751647, rs761796768, rs761828482, rs761859611,rs761882804, rs761913009, rs761914605, rs761934425, rs761968706,rs762006599, rs762125685, rs762211580, rs762226529, rs762283787,rs762303998, rs762483687, rs762546467, rs762570628, rs762582573,rs762618243, rs762627114, rs762771301, rs762876977, rs762943482,rs762981013, rs763034153, rs763047690, rs763099634, rs763214827,rs763214846, rs763350634, rs763362455, rs763381837, rs763389143,rs763444638, rs763497680, rs763522643, rs763568920, rs763608460,rs763697863, rs763712712, rs763774686, rs763799379, rs763851107,rs763859635, rs763890649, rs763907879, rs763914758, rs763945017,rs764046799, rs764072081, rs764099561, rs764174190, rs764280512,rs764286466, rs764334726, rs764336613, rs764439180, rs764473740,rs764522054, rs764579879, rs764591974, rs764680965, rs764715821,rs764749298, rs764794547, rs764858224, rs764905877, rs764907748,rs764954235, rs764991426, rs765048134, rs765051553, rs765079532,rs765119055, rs765174065, rs765189820, rs765235904, rs765243245,rs765266637, rs765331376, rs765350640, rs765369504, rs765431582,rs765432253, rs765439946, rs765463082, rs765520038, rs765682027,rs765774591, rs765812814, rs765886731, rs765906787, rs765910207,rs765914203, rs765926917, rs765964805, rs766104084, rs766125236,rs766286225, rs766338743, rs766418873, rs766418901, rs766422289,rs766482364, rs766557426, rs766570845, rs766640359, rs766646217,rs766822218, rs766828774, rs766837655, rs766859707, rs766944930,rs766981529, rs767009640, rs767033042, rs767060337, rs767214355,rs767258249, rs767305032, rs767312106, rs767360120, rs767392013,rs767561967, rs767768129, rs767771897, rs767788419, rs767799735,rs767800023, rs768080447, rs768083210, rs768206746, rs768284541,rs768365237, rs768390959, rs768417052, rs768465375, rs768538403,rs768597548, rs768706642, rs768812125, rs768813846, rs768915855,rs768994065, rs769017393, rs769018282, rs769023061, rs769024262,rs769069042, rs769129492, rs769214729, rs769217873, rs769219806,rs769385677, rs769551807, rs769597716, rs769622690, rs769648456,rs769731197, rs769749617, rs769801596, rs769809024, rs769818541,rs769964799, rs770016889, rs770035607, rs770045903, rs770119513,rs770148893, rs770164381, rs770181308, rs770255396, rs770266116,rs770358984, rs770374274, rs770484034, rs770512665, rs770587800,rs770719238, rs770740012, rs770747229, rs770786181, rs770873775,rs770915628, rs770989683, rs771077016, rs771080028, rs771112679,rs771139931, rs771232307, rs771248299, rs771261012, rs771288323,rs771338362, rs771406749, rs771426657, rs771554986, rs771641368,rs771645454, rs771698726, rs771734877, rs771741738, rs771764405,rs771769796, rs771806866, rs771962267, rs772051916, rs772112577,rs772165419, rs772221900, rs772228903, rs772230274, rs772235737,rs772242251, rs772299197, rs772311336, rs772442561, rs772444228,rs772504493, rs772514611, rs772640099, rs772765835, rs772792838,rs772808446, rs772890193, rs772986241, rs773030462, rs773037460,rs773078701, rs773211045, rs773305880, rs773307918, rs773327031,rs773348446, rs773394059, rs773403637, rs773415492, rs773475926,rs773516984, rs773563136, rs773787822, rs773808646, rs773811368,rs773849840, rs773886266, rs773905916, rs774012066, rs774207880,rs774229738, rs774240595, rs774420737, rs774430952, rs774554121,rs774560137, rs774564791, rs774569101, rs774588330, rs774615995,rs774688311, rs774767443, rs774868695, rs774903867, rs774923254,rs774925684, rs774934785, rs774980016, rs775007490, rs775044013,rs775105018, rs775192787, rs775203380, rs775228379, rs775258389,rs775299640, rs775339427, rs775394437, rs775415473, rs775550206,rs775635196, rs775689438, rs775738104, rs775825051, rs775878558,rs775878637, rs776042543, rs776073853, rs776083430, rs776152947,rs776276813, rs776278329, rs776298636, rs776418340, rs776439113,rs776470314, rs776563297, rs776624188, rs776680792, rs776737538,rs776745208, rs776773228, rs776783496, rs776785502, rs776817542,rs776826689, rs776845104, rs776893288, rs776947384, rs776974102,rs777075412, rs777136217, rs777208445, rs777306136, rs777336060,rs777397193, rs777406730, rs777432099, rs777436847, rs777539780,rs777662187, rs777697046, rs777699097, rs777700238, rs777727222,rs777758286, rs777766926, rs777892234, rs777954711, rs777997008,rs778040387, rs778107598, rs778177112, rs778378811, rs778615261,rs778664525, rs778719428, rs778736663, rs778752848, rs778875311,rs778909991, rs779017511, rs779132852, rs779224998, rs779231833,rs779241028, rs779246332, rs779286579, rs779327088, rs779428964,rs779431480, rs779480027, rs779602583, rs779666069, rs779707457,rs779735498, rs779783880, rs779788849, rs779855597, rs779870173,rs779924630, rs780014587, rs780072959, rs780088411, rs780219814,rs780266997, rs780323328, rs780368194, rs780386793, rs780391826,rs780426461, rs780449707, rs780510809, rs780533501, rs780537281,rs780542223, rs780546534, rs780550917, rs780687906, rs780834162,rs780858451, rs780880744, rs780953815, rs780955201, rs780965711,rs781010216, rs781017028, rs781078843, rs781114491, rs781136461,rs781224455, rs781230982, rs781409321, rs781411576, rs781514479,rs781534991, rs781635721, rs781660401, rs781678909, rs781682041,rs781725943, VAR_003437, VAR_009129, VAR_009130, VAR_009131, VAR_009134,VAR_009135, VAR_015373, VAR_023770, VAR_023771, VAR_042230, VAR_042231,VAR_067170, VAR_067171, VAR_067172, VAR_067174, VAR_067175, VAR_067176,VAR_067177, VAR_067178, VAR_067179, VAR_067180, VAR_067181, VAR_067182,VAR_071605, VAR_071606, VAR_071607, and VAR_071608.

There are various mutations associated with CORD, which can beinsertions, deletions, missense, nonsense, frameshift and othermutations, with the common effect of inactivating the GUCY2D gene. Anyone or more of the mutations can be repaired to restore RetGC1 proteinactivity. For example, the pathological variants, R838H, R838C, orR838S, can be restored or corrected (See Table 3).

TABLE 3 Variant Location Variant type R838H 8,014,701 missense R838C8,014,700 missense R838S 8,014,700 missense

Exon Deletion

Another genome engineering strategy involves exon deletion. Targeteddeletion of specific exons can be an attractive strategy for treating alarge subset of patients with a single therapeutic cocktail. Deletionscan either be single exon deletions or multi-exon deletions. Whilemulti-exon deletions can reach a larger number of patients, for largerdeletions the efficiency of deletion greatly decreases with increasedsize. Therefore, deletions range can be from 40 to 10,000 base pairs(bp) in size. For example, deletions can range from 40-100; 100-300;300-500; 500-1,000; 1,000-2,000; 2,000-3,000; 3,000-5,000; or5,000-10,000 base pairs in size.

As stated previously, the GUCY2D gene contains 20 exons. Any one or moreof the 20 exons can contain a mutation. Any one or more of the 20mutated exons, or aberrant intronic splice acceptor or donor sites, canbe deleted to restore or partially restore the GUCY2D function. In someexamples, the methods provide gRNA pairs that can be used to delete anyone or more of the mutated exons 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, or any combinations thereof.

To ensure that the pre-mRNA is properly processed following deletion,the surrounding splicing signals can be deleted. Splicing donor andacceptors are generally within 100 base pairs of the neighboring intron.Therefore, in some examples, methods can provide all gRNAs that cutapproximately +/−100-3100 bp with respect to each exon/intron junctionof interest.

For any of the genome editing strategies, gene editing can be confirmedby sequencing or PCR analysis.

In Vivo Based Therapy

Provided herein are methods for treating a patient with autosomaldominant CORD. In some aspects, the method is an in vivo cell-basedtherapy. Chromosomal DNA of the cells in the autosomal dominant CORDpatient can be edited using the materials and methods described herein.For example, the in vivo method can comprise editing a R838H, R838C, orR838S mutation in a GUCY2D gene in a cell of a patient, such asphotoreceptor cells or retinal progenitor cells.

Although certain cells present an attractive target for ex vivotreatment and therapy, increased efficacy in delivery may permit directin vivo delivery to such cells. Ideally the targeting and editing wouldbe directed to the relevant cells. Cleavage in other cells can also beprevented by the use of promoters only active in certain cells and ordevelopmental stages. Additional promoters are inducible, and thereforecan be temporally controlled if the nuclease is delivered as a plasmid.The amount of time that delivered RNA and protein remain in the cell canalso be adjusted using treatments or domains added to change thehalf-life. In vivo treatment would eliminate a number of treatmentsteps, but a lower rate of delivery can require higher rates of editing.In vivo treatment can eliminate problems and losses from ex vivotreatment and engraftment.

An advantage of in vivo gene therapy can be the ease of therapeuticproduction and administration. The same therapeutic approach and therapywill have the potential to be used to treat more than one patient, forexample a number of patients who share the same or similar genotype orallele. In contrast, ex vivo cell therapy typically requires using apatient's own cells, which are isolated, manipulated and returned to thesame patient.

Ex Vivo Based Therapy

Provided herein are methods for treating a patient with autosomaldominant CORD. An aspect of such method is an ex vivo cell-basedtherapy. For example, a patient-specific induced pluripotent stem cell(iPSC) can be created. Then, the chromosomal DNA of these iPSC cells canbe edited using the materials and methods described herein. For example,the method can comprise editing within or near a R838H, R838C, or R838Smutation in the GUCY2D gene of the iPSC. Next, the genome-edited iPSCscan be differentiated into other cells, such as photoreceptor cells orretinal progenitor cells. Finally, the differentiated cells, such asphotoreceptor cell or retinal progenitor cell, can be implanted into thepatient.

Another aspect of such method is an ex vivo cell-based therapy. Forexample, photoreceptor cells or retinal progenitor cells can be isolatedfrom the patient. Next, the chromosomal DNA of these photoreceptor cellsor retinal progenitor cells can be edited using the materials andmethods described herein. For example, the method can comprise editingwithin or near a R838H, R838C, or R838S mutation in the GUCY2D gene ofthe photoreceptor cells or retinal progenitor cells. Finally, thegenome-edited photoreceptor cells or retinal progenitor cells can beimplanted into the patient.

Another aspect of such method is an ex vivo cell-based therapy. Forexample, a mesenchymal stem cell can be isolated from the patient, whichcan be isolated from the patient's bone marrow or peripheral blood.Next, the chromosomal DNA of these mesenchymal stem cells can be editedusing the materials and methods described herein. For example, themethod can comprise editing within or near a R838H, R838C, or R838Smutation in the GUCY2D gene of the mesenchymal stem cells. Next, thegenome-edited mesenchymal stem cells can be differentiated into any typeof cell, e.g., photoreceptor cells or retinal progenitor cells. Finally,the differentiated cells, e.g., photoreceptor cells or retinalprogenitor cells can be implanted into the patient.

One advantage of an ex vivo cell therapy approach is the ability toconduct a comprehensive analysis of the therapeutic prior toadministration. Nuclease-based therapeutics can have some level ofoff-target effects. Performing gene correction ex vivo allows one tocharacterize the corrected cell population prior to implantation. Thepresent disclosure includes sequencing the entire genome of thecorrected cells to ensure that the off-target effects, if any, can be ingenomic locations associated with minimal risk to the patient.Furthermore, populations of specific cells, including clonalpopulations, can be isolated prior to implantation.

Another advantage of ex vivo cell therapy relates to genetic correctionin iPSCs compared to other primary cell sources. iPSCs are prolific,making it easy to obtain the large number of cells that will be requiredfor a cell-based therapy. Furthermore, iPSCs are an ideal cell type forperforming clonal isolations. This allows screening for the correctgenomic correction, without risking a decrease in viability. Incontrast, other primary cells, such as photoreceptor cells or retinalprogenitor cells, are viable for only a few passages and difficult toclonally expand. Thus, manipulation of iPSCs for the treatment ofautosomal dominant CORD can be much easier, and can shorten the amountof time needed to make the desired genetic correction.

Genome Editing

Genome editing refers to the process of modifying the nucleotidesequence of a genome, such as in a precise or pre-determined manner.Examples of methods of genome editing described herein include methodsof using site-directed nucleases to cut DNA at precise target locationsin the genome, thereby creating single-strand or double-strand DNAbreaks at particular locations within the genome. Such breaks can be andregularly are repaired by natural, endogenous cellular processes, suchas HDR and NHEJ. These two main DNA repair processes consist of a familyof alternative pathways. NHEJ directly joins the DNA ends resulting froma double-strand break, sometimes with the loss or addition of nucleotidesequence, which may disrupt or enhance gene expression. HDR utilizes ahomologous sequence, or donor sequence, as a template for inserting adefined DNA sequence at the break point. The homologous sequence can bein the endogenous genome, such as a sister chromatid. Alternatively, thedonor can be an exogenous nucleic acid, such as a plasmid, asingle-strand oligonucleotide, a double-stranded oligonucleotide, aduplex oligonucleotide or a virus, that has regions of high homologywith the nuclease-cleaved locus, but which can also contain additionalsequence or sequence changes including deletions that can beincorporated into the cleaved target locus. A third repair mechanism canbe microhomology-mediated end joining (MMEJ), also referred to as“Alternative NHEJ (ANHEJ)”, in which the genetic outcome is similar toNHEJ in that small deletions and insertions can occur at the cleavagesite. MMEJ can make use of homologous sequences of a few base pairsflanking the DNA break site to drive a more favored DNA end joiningrepair outcome, and recent reports have further elucidated the molecularmechanism of this process. In some instances, it may be possible topredict likely repair outcomes based on analysis of potentialmicrohomologies at the site of the DNA break.

Each of these genome editing mechanisms can be used to create desiredgenomic alterations. A step in the genome editing process can be tocreate one or two DNA breaks, the latter as double-strand breaks or astwo single-stranded breaks, in the target locus as near the site ofintended mutation. This can be achieved via the use of site-directedpolypeptides, as described and illustrated herein.

Site-directed polypeptides, such as a DNA endonuclease, can introducedouble-strand breaks or single-strand breaks in nucleic acids, e.g.,genomic DNA. The double-strand break can stimulate a cell's endogenousDNA-repair pathways [e.g., homology-dependent repair (HDR) ornon-homologous end joining (NHEJ) or (ANHEJ) or (MMEJ)]. NHEJ can repaircleaved target nucleic acid without the need for a homologous template.This can sometimes result in small deletions or insertions (indels) inthe target nucleic acid at the site of cleavage, and can lead todisruption or alteration of gene expression. The deletions range can befrom 40 to 10,000 base pairs (bp) in size. For example, deletions canrange from 40-100; 100-300; 300-500; 500-1,000; 1,000-2,000;2,000-3,000; 3,000-5,000; or 5,000-10,000 base pairs in size. HDR canoccur when a homologous repair template, or donor, is available. Thehomologous donor template can comprise at least a portion of thewild-type GUCY2D gene, or cDNA. The at least a portion of the wild-typeGUCY2D gene or cDNA can be exon 1, exon 2, exon 3, exon 4, exon 5, exon6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14,exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, intronic regions,fragments or combinations thereof, or the enitre GUCY2D gene or cDNA.The donor template can be either a single or double strandedpolynucleotide. The donor template can be up to 5 KB. The donor templatecan be up to 4 KB. The donor template can be up to 3 KB. The donortemplate can be up to 2 KB. The donor template can be up to 1 KB. Thedonor template can be delivered by AAV. The homologous donor templatecan comprise sequences that can be homologous to sequences flanking thetarget nucleic acid cleavage site. For example, the donor template canhave homologous arms to the 17p13.1 region. The donor template can alsohave homologous arms to the pathological variant R838H, R838C, or R838S.The sister chromatid can be used by the cell as the repair template.However, for the purposes of genome editing, the repair template can besupplied as an exogenous nucleic acid, such as a plasmid, duplexoligonucleotide, single-strand oligonucleotide, double-strandedoligonucleotide, or viral nucleic acid. With exogenous donor templates,an additional nucleic acid sequence (such as a transgene) ormodification (such as a single or multiple base change or a deletion)can be introduced between the flanking regions of homology so that theadditional or altered nucleic acid sequence also becomes incorporatedinto the target locus. MMEJ can result in a genetic outcome that issimilar to NHEJ, in that small deletions and insertions can occur at thecleavage site. MMEJ can make use of homologous sequences of a few basepairs flanking the cleavage site to drive a favored end-joining DNArepair outcome. In some instances, it may be possible to predict likelyrepair outcomes based on analysis of potential microhomologies in thenuclease target regions.

Thus, in some cases, homologous recombination can be used to insert anexogenous polynucleotide sequence into the target nucleic acid cleavagesite. An exogenous polynucleotide sequence is termed a donorpolynucleotide (or donor or donor sequence or polynucleotide donortemplate) herein. The donor polynucleotide, a portion of the donorpolynucleotide, a copy of the donor polynucleotide, or a portion of acopy of the donor polynucleotide can be inserted into the target nucleicacid cleavage site. The donor polynucleotide can be an exogenouspolynucleotide sequence, i.e., a sequence that does not naturally occurat the target nucleic acid cleavage site.

The modifications of the target DNA due to NHEJ and/or HDR can lead to,for example, mutations, deletions, alterations, integrations, genecorrection, gene replacement, gene tagging, transgene insertion,nucleotide deletion, gene disruption, translocations and/or genemutation. The processes of deleting genomic DNA and integratingnon-native nucleic acid into genomic DNA are examples of genome editing.

CRISPR Endonuclease System

A CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)genomic locus can be found in the genomes of many prokaryotes (e.g.,bacteria and archaea). In prokaryotes, the CRISPR locus encodes productsthat function as a type of immune system to help defend the prokaryotesagainst foreign invaders, such as virus and phage. There are threestages of CRISPR locus function: integration of new sequences into theCRISPR locus, expression of CRISPR RNA (crRNA), and silencing of foreigninvader nucleic acid. Five types of CRISPR systems (e.g., Type I, TypeII, Type III, Type U, and Type V) have been identified.

A CRISPR locus includes a number of short repeating sequences referredto as “repeats.” When expressed, the repeats can form secondarystructures (e.g., hairpins) and/or comprise unstructured single-strandedsequences. The repeats usually occur in clusters and frequently divergebetween species. The repeats are regularly interspaced with uniqueintervening sequences referred to as “spacers,” resulting in arepeat-spacer-repeat locus architecture. The spacers are identical to orhave high homology with known foreign invader sequences. A spacer-repeatunit encodes a crisprRNA (crRNA), which is processed into a mature formof the spacer-repeat unit. A crRNA comprises a “seed” or spacer sequencethat is involved in targeting a target nucleic acid (in the naturallyoccurring form in prokaryotes, the spacer sequence targets the foreigninvader nucleic acid). A spacer sequence is located at the 5′ or 3′ endof the crRNA.

A CRISPR locus also comprises polynucleotide sequences encoding CRISPRAssociated (Cas) genes. Cas genes encode endonucleases involved in thebiogenesis and the interference stages of crRNA function in prokaryotes.Some Cas genes comprise homologous secondary and/or tertiary structures.

Type II CRISPR Systems

crRNA biogenesis in a Type II CRISPR system in nature requires atrans-activating CRISPR RNA (tracrRNA). The tracrRNA can be modified byendogenous RNaseIII, and then hybridizes to a crRNA repeat in thepre-crRNA array. Endogenous RNaseIII can be recruited to cleave thepre-crRNA. Cleaved crRNAs can be subjected to exoribonuclease trimmingto produce the mature crRNA form (e.g., 5′ trimming). The tracrRNA canremain hybridized to the crRNA, and the tracrRNA and the crRNA associatewith a site-directed polypeptide (e.g., Cas9). The crRNA of thecrRNA-tracrRNA-Cas9 complex can guide the complex to a target nucleicacid to which the crRNA can hybridize. Hybridization of the crRNA to thetarget nucleic acid can activate Cas9 for targeted nucleic acidcleavage. The target nucleic acid in a Type II CRISPR system is referredto as a protospacer adjacent motif (PAM). In nature, the PAM isessential to facilitate binding of a site-directed polypeptide (e.g.,Cas9) to the target nucleic acid. Type II systems (also referred to asNmeni or CASS4) are further subdivided into Type II-A (CASS4) and II-B(CASS4a). Jinek et al., Science, 337(6096):816-821 (2012) showed thatthe CRISPR/Cas9 system is useful for RNA-programmable genome editing,and international patent application publication number WO2013/176772provides numerous examples and applications of the CRISPR/Casendonuclease system for site-specific gene editing.

Type V CRISPR Systems

Type V CRISPR systems have several important differences from Type IIsystems. For example, Cpf1 is a single RNA-guided endonuclease that, incontrast to Type II systems, lacks tracrRNA. In fact, Cpf1-associatedCRISPR arrays can be processed into mature crRNAs without therequirement of an additional trans-activating tracrRNA. The Type VCRISPR array can be processed into short mature crRNAs of 42-44nucleotides in length, with each mature crRNA beginning with 19nucleotides of direct repeat followed by 23-25 nucleotides of spacersequence. In contrast, mature crRNAs in Type II systems can start with20-24 nucleotides of spacer sequence followed by about 22 nucleotides ofdirect repeat. Also, Cpf1 can utilize a T-rich protospacer-adjacentmotif such that Cpf1-crRNA complexes efficiently cleave target DNApreceded by a short T-rich PAM, which is in contrast to the G-rich PAMfollowing the target DNA for Type II systems. Thus, Type V systemscleave at a point that is distant from the PAM, while Type II systemscleave at a point that is adjacent to the PAM. In addition, in contrastto Type II systems, Cpf1 cleaves DNA via a staggered DNA double-strandedbreak with a 4 or 5 nucleotide 5′ overhang. Type II systems cleave via ablunt double-stranded break. Similar to Type II systems, Cpf1 contains apredicted RuvC-like endonuclease domain, but lacks a second HNHendonuclease domain, which is in contrast to Type II systems.

Cas Genes/Polypeptides and Protospacer Adjacent Motifs

Exemplary CRISPR/Cas polypeptides include the Cas9 polypeptides in FIG.1 of Fonfara et al., Nucleic Acids Research, 42: 2577-2590 (2014). TheCRISPR/Cas gene naming system has undergone extensive rewriting sincethe Cas genes were discovered. FIG. 5 of Fonfara, supra, provides PAMsequences for the Cas9 polypeptides from various species.

Site-Directed Polypeptides

A site-directed polypeptide is a nuclease used in genome editing tocleave DNA and/or induce site-directed mutagenesis. The site-directednuclease can be administered to a cell or a patient as either: one ormore polypeptides, or one or more mRNAs encoding the polypeptide. Any ofthe enzymes or orthologs listed in SEQ ID NOs. 1-612, or disclosedherein, can be utilized in the methods herein.

In the context of a CRISPR/Cas or CRISPR/Cpf1 system, the site-directedpolypeptide can bind to a guide RNA that, in turn, specifies the site inthe target DNA to which the polypeptide is directed. In the CRISPR/Casor CRISPR/Cpf1 systems disclosed herein, the site-directed polypeptidecan be an endonuclease, such as a DNA endonuclease.

A site-directed polypeptide can comprise a plurality of nucleicacid-cleaving (i.e., nuclease) domains. Two or more nucleicacid-cleaving domains can be linked together via a linker. For example,the linker can comprise a flexible linker. Linkers can comprise 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 30, 35, 40 or more amino acids in length.

Naturally-occurring wild-type Cas9 enzymes comprise two nucleasedomains, a HNH nuclease domain and a RuvC domain. Herein, the “Cas9”refers to both naturally occurring and recombinant Cas9s. Cas9 enzymescontemplated herein can comprise a HNH or HNH-like nuclease domain,and/or a RuvC or RuvC-like nuclease domain.

HNH or HNH-like domains comprise a McrA-like fold. HNH or HNH-likedomains comprises two antiparallel β-strands and an α-helix. HNH orHNH-like domains comprises a metal binding site (e.g., a divalent cationbinding site). HNH or HNH-like domains can cleave one strand of a targetnucleic acid (e.g., the complementary strand of the crRNA targetedstrand).

RuvC or RuvC-like domains comprise an RNaseH or RNaseH-like fold.RuvC/RNaseH domains are involved in a diverse set of nucleic acid-basedfunctions including acting on both RNA and DNA. The RNaseH domaincomprises 5 β-strands surrounded by a plurality of α-helices.RuvC/RNaseH or RuvC/RNaseH-like domains comprise a metal binding site(e.g., a divalent cation binding site). RuvC/RNaseH or RuvC/RNaseH-likedomains can cleave one strand of a target nucleic acid (e.g., thenon-complementary strand of a double-stranded target DNA).

Site-directed polypeptides can introduce double-strand breaks orsingle-strand breaks in nucleic acids, e.g., genomic DNA. Thedouble-strand break can stimulate a cell's endogenous DNA-repairpathways (e.g., HDR or NHEJ or ANHEJ or MMEJ). NHEJ can repair cleavedtarget nucleic acid without the need for a homologous template. This cansometimes result in small deletions or insertions (indels) in the targetnucleic acid at the site of cleavage, and can lead to disruption oralteration of gene expression. HDR can occur when a homologous repairtemplate, or donor, is available. The homologous donor template cancomprise sequences that are homologous to sequences flanking the targetnucleic acid cleavage site. The sister chromatid can be used by the cellas the repair template. However, for the purposes of genome editing, therepair template can be supplied as an exogenous nucleic acid, such as aplasmid, duplex oligonucleotide, single-strand oligonucleotide or viralnucleic acid. With exogenous donor templates, an additional nucleic acidsequence (such as a transgene) or modification (such as a single ormultiple base change or a deletion) can be introduced between theflanking regions of homology so that the additional or altered nucleicacid sequence also becomes incorporated into the target locus. MMEJ canresult in a genetic outcome that is similar to NHEJ, in that smalldeletions and insertions can occur at the cleavage site. MMEJ can makeuse of homologous sequences of a few base pairs flanking the cleavagesite to drive a favored end-joining DNA repair outcome. In someinstances, it may be possible to predict likely repair outcomes based onanalysis of potential microhomologies in the nuclease target regions.

Thus, in some cases, homologous recombination can be used to insert anexogenous polynucleotide sequence into the target nucleic acid cleavagesite. An exogenous polynucleotide sequence is termed a donorpolynucleotide (or donor or donor sequence) herein. The donorpolynucleotide, a portion of the donor polynucleotide, a copy of thedonor polynucleotide, or a portion of a copy of the donor polynucleotidecan be inserted into the target nucleic acid cleavage site. The donorpolynucleotide can be an exogenous polynucleotide sequence, i.e., asequence that does not naturally occur at the target nucleic acidcleavage site.

The site-directed polypeptide can comprise an amino acid sequence havingat least 10%, at least 15%, at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 99%, or 100% amino acidsequence identity to a wild-type exemplary site-directed polypeptide[e.g., Cas9 from S. pyogenes, US2014/0068797 Sequence ID No. 8 orSapranauskas et al., Nucleic Acids Res, 39(21): 9275-9282 (2011)], andvarious other site-directed polypeptides. The site-directed polypeptidecan comprise at least 70, 75, 80, 85, 90, 95, 97, 99, or 100% identityto a wild-type site-directed polypeptide (e.g., Cas9 from S. pyogenes,supra) over 10 contiguous amino acids. The site-directed polypeptide cancomprise at most: 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to awild-type site-directed polypeptide (e.g., Cas9 from S. pyogenes, supra)over 10 contiguous amino acids. The site-directed polypeptide cancomprise at least: 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to awild-type site-directed polypeptide (e.g., Cas9 from S. pyogenes, supra)over 10 contiguous amino acids in a HNH nuclease domain of thesite-directed polypeptide. The site-directed polypeptide can comprise atmost: 70, 75, 80, 85, 90, 95, 97, 99, or 100% identity to a wild-typesite-directed polypeptide (e.g., Cas9 from S. pyogenes, supra) over 10contiguous amino acids in a HNH nuclease domain of the site-directedpolypeptide. The site-directed polypeptide can comprise at least: 70,75, 80, 85, 90, 95, 97, 99, or 100% identity to a wild-typesite-directed polypeptide (e.g., Cas9 from S. pyogenes, supra) over 10contiguous amino acids in a RuvC nuclease domain of the site-directedpolypeptide. The site-directed polypeptide can comprise at most: 70, 75,80, 85, 90, 95, 97, 99, or 100% identity to a wild-type site-directedpolypeptide (e.g., Cas9 from S. pyogenes, supra) over 10 contiguousamino acids in a RuvC nuclease domain of the site-directed polypeptide.

The site-directed polypeptide can comprise a modified form of awild-type exemplary site-directed polypeptide. The modified form of thewild-type exemplary site-directed polypeptide can comprise a mutationthat reduces the nucleic acid-cleaving activity of the site-directedpolypeptide. The modified form of the wild-type exemplary site-directedpolypeptide can have less than 90%, less than 80%, less than 70%, lessthan 60%, less than 50%, less than 40%, less than 30%, less than 20%,less than 10%, less than 5%, or less than 1% of the nucleicacid-cleaving activity of the wild-type exemplary site-directedpolypeptide (e.g., Cas9 from S. pyogenes, supra). The modified form ofthe site-directed polypeptide can have no substantial nucleicacid-cleaving activity. When a site-directed polypeptide is a modifiedform that has no substantial nucleic acid-cleaving activity, it isreferred to herein as “enzymatically inactive.”

The modified form of the site-directed polypeptide can comprise amutation such that it can induce a SSB on a target nucleic acid (e.g.,by cutting only one of the sugar-phosphate backbones of a double-strandtarget nucleic acid). The mutation can result in less than 90%, lessthan 80%, less than 70%, less than 60%, less than 50%, less than 40%,less than 30%, less than 20%, less than 10%, less than 5%, or less than1% of the nucleic acid-cleaving activity in one or more of the pluralityof nucleic acid-cleaving domains of the wild-type site directedpolypeptide (e.g., Cas9 from S. pyogenes, supra). The mutation canresult in one or more of the plurality of nucleic acid-cleaving domainsretaining the ability to cleave the complementary strand of the targetnucleic acid, but reducing its ability to cleave the non-complementarystrand of the target nucleic acid. The mutation can result in one ormore of the plurality of nucleic acid-cleaving domains retaining theability to cleave the non-complementary strand of the target nucleicacid, but reducing its ability to cleave the complementary strand of thetarget nucleic acid. For example, residues in the wild-type exemplary S.pyogenes Cas9 polypeptide, such as Asp10, His840, Asn854 and Asn856, aremutated to inactivate one or more of the plurality of nucleicacid-cleaving domains (e.g., nuclease domains). The residues to bemutated can correspond to residues Asp10, His840, Asn854 and Asn856 inthe wild-type exemplary S. pyogenes Cas9 polypeptide (e.g., asdetermined by sequence and/or structural alignment). Non-limitingexamples of mutations include D10A, H840A, N854A or N856A. Mutationsother than alanine substitutions can be suitable.

A D10A mutation can be combined with one or more of H840A, N854A, orN856A mutations to produce a site-directed polypeptide substantiallylacking DNA cleavage activity. A H840A mutation can be combined with oneor more of D10A, N854A, or N856A mutations to produce a site-directedpolypeptide substantially lacking DNA cleavage activity. A N854Amutation can be combined with one or more of H840A, D10A, or N856Amutations to produce a site-directed polypeptide substantially lackingDNA cleavage activity. A N856A mutation can be combined with one or moreof H840A, N854A, or D10A mutations to produce a site-directedpolypeptide substantially lacking DNA cleavage activity. Site-directedpolypeptides that comprise one substantially inactive nuclease domainare referred to as “nickases”.

Nickase variants of RNA-guided endonucleases, for example Cas9, can beused to increase the specificity of CRISPR-mediated genome editing. Wildtype Cas9 is typically guided by a single guide RNA designed tohybridize with a specified ˜20 nucleotide sequence in the targetsequence (such as an endogenous genomic locus). However, severalmismatches can be tolerated between the guide RNA and the target locus,effectively reducing the length of required homology in the target siteto, for example, as little as 13 nt of homology, and thereby resultingin elevated potential for binding and double-strand nucleic acidcleavage by the CRISPR/Cas9 complex elsewhere in the target genome—alsoknown as off-target cleavage. Because nickase variants of Cas9 each onlycut one strand, in order to create a double-strand break it is necessaryfor a pair of nickases to bind in close proximity and on oppositestrands of the target nucleic acid, thereby creating a pair of nicks,which is the equivalent of a double-strand break. This requires that twoseparate guide RNAs—one for each nickase—must bind in close proximityand on opposite strands of the target nucleic acid. This requirementessentially doubles the minimum length of homology needed for thedouble-strand break to occur, thereby reducing the likelihood that adouble-strand cleavage event will occur elsewhere in the genome, wherethe two guide RNA sites—if they exist—are unlikely to be sufficientlyclose to each other to enable the double-strand break to form. Asdescribed in the art, nickases can also be used to promote HDR versusNHEJ. HDR can be used to introduce selected changes into target sites inthe genome through the use of specific donor sequences that effectivelymediate the desired changes.

Mutations contemplated can include substitutions, additions, anddeletions, or any combination thereof. The mutation converts the mutatedamino acid to alanine. The mutation converts the mutated amino acid toanother amino acid (e.g., glycine, serine, threonine, cysteine, valine,leucine, isoleucine, methionine, proline, phenylalanine, tyrosine,tryptophan, aspartic acid, glutamic acid, asparagines, glutamine,histidine, lysine, or arginine). The mutation converts the mutated aminoacid to a non-natural amino acid (e.g., selenomethionine). The mutationconverts the mutated amino acid to amino acid mimics (e.g.,phosphomimics). The mutation can be a conservative mutation. Forexample, the mutation converts the mutated amino acid to amino acidsthat resemble the size, shape, charge, polarity, conformation, and/orrotamers of the mutated amino acids (e.g., cysteine/serine mutation,lysine/asparagine mutation, histidine/phenylalanine mutation). Themutation can cause a shift in reading frame and/or the creation of apremature stop codon. Mutations can cause changes to regulatory regionsof genes or loci that affect expression of one or more genes.

The site-directed polypeptide (e.g., variant, mutated, enzymaticallyinactive and/or conditionally enzymatically inactive site-directedpolypeptide) can target nucleic acid. The site-directed polypeptide(e.g., variant, mutated, enzymatically inactive and/or conditionallyenzymatically inactive endoribonuclease) can target DNA. Thesite-directed polypeptide (e.g., variant, mutated, enzymaticallyinactive and/or conditionally enzymatically inactive endoribonuclease)can target RNA.

The site-directed polypeptide can comprise one or more non-nativesequences (e.g., the site-directed polypeptide is a fusion protein).

The site-directed polypeptide can comprise an amino acid sequencecomprising at least 15% amino acid identity to a Cas9 from a bacterium(e.g., S. pyogenes), a nucleic acid binding domain, and two nucleic acidcleaving domains (i.e., a HNH domain and a RuvC domain).

The site-directed polypeptide can comprise an amino acid sequencecomprising at least 15% amino acid identity to a Cas9 from a bacterium(e.g., S. pyogenes), and two nucleic acid cleaving domains (i.e., a HNHdomain and a RuvC domain).

The site-directed polypeptide can comprise an amino acid sequencecomprising at least 15% amino acid identity to a Cas9 from a bacterium(e.g., S. pyogenes), and two nucleic acid cleaving domains, wherein oneor both of the nucleic acid cleaving domains comprise at least 50% aminoacid identity to a nuclease domain from Cas9 from a bacterium (e.g., S.pyogenes).

The site-directed polypeptide can comprise an amino acid sequencecomprising at least 15% amino acid identity to a Cas9 from a bacterium(e.g., S. pyogenes), two nucleic acid cleaving domains (i.e., a HNHdomain and a RuvC domain), and non-native sequence (for example, anuclear localization signal) or a linker linking the site-directedpolypeptide to a non-native sequence.

The site-directed polypeptide can comprise an amino acid sequencecomprising at least 15% amino acid identity to a Cas9 from a bacterium(e.g., S. pyogenes), two nucleic acid cleaving domains (i.e., a HNHdomain and a RuvC domain), wherein the site-directed polypeptidecomprises a mutation in one or both of the nucleic acid cleaving domainsthat reduces the cleaving activity of the nuclease domains by at least50%.

The site-directed polypeptide can comprise an amino acid sequencecomprising at least 15% amino acid identity to a Cas9 from a bacterium(e.g., S. pyogenes), and two nucleic acid cleaving domains (i.e., a HNHdomain and a RuvC domain), wherein one of the nuclease domains comprisesmutation of aspartic acid 10, and/or wherein one of the nuclease domainscan comprise a mutation of histidine 840, and wherein the mutationreduces the cleaving activity of the nuclease domain(s) by at least 50%.

The one or more site-directed polypeptides, e.g. DNA endonucleases, cancomprise two nickases that together effect one double-strand break at aspecific locus in the genome, or four nickases that together effect orcause two double-strand breaks at specific loci in the genome.Alternatively, one site-directed polypeptide, e.g. DNA endonuclease, caneffect or cause one double-strand break at a specific locus in thegenome.

Non-limiting examples of Cas9 orthologs from other bacterial strainsincluding but not limited to, Cas proteins identified in Acaryochlorismarina MBIC11017; Acetohalobium arabaticum DSM 5501; Acidithiobacilluscaldus; Acidithiobacillus ferrooxidans ATCC 23270; Alicyclobacillusacidocaldarius LAA1; Alicyclobacillus acidocaldarius subsp.acidocaldarius DSM 446; Allochromatium vinosum DSM 180; Ammonifexdegensii KC4; Anabaena variabilis ATCC 29413; Arthrospira maxima CS-328;Arthrospira platensis str. Paraca; Arthrospira sp. PCC 8005; Bacilluspseudomycoides DSM 12442; Bacillus selenitireducens MLS10;Burkholderiales bacterium 1_1_47; Caldicelulosiruptor becscii DSM 6725;Candidatus Desulforudis audaxviator MP104C; Caldicellulosiruptorhydrothermalis_108; Clostridium phage c-st; Clostridium botulinum A3str. Loch Maree; Clostridium botulinum Ba4 str. 657; Clostridiumdifficile QCD-63q42; Crocosphaera watsonii WH 8501; Cyanothece sp. ATCC51142; Cyanothece sp. CCY0110; Cyanothece sp. PCC 7424; Cyanothece sp.PCC 7822; Exiguobacterium sibiricum 255-15; Finegoldia magna ATCC 29328;Ktedonobacter racemifer DSM 44963; Lactobacillus delbrueckii subsp.bulgaricus PB2003/044-T3-4; Lactobacillus salivarius ATCC 11741;Listeria innocua; Lyngbya sp. PCC 8106; Marinobacter sp. ELB17;Methanohalobium evestigatum Z-7303; Microcystis phage Ma-LMM01;Microcystis aeruginosa NIES-843; Microscilla marina ATCC 23134;Microcoleus chthonoplastes PCC 7420; Neisseria meningitidis;Nitrosococcus halophilus Nc4; Nocardiopsis dassonvillei subsp.dassonvillei DSM 43111; Nodularia spumigena CCY9414; Nostoc sp. PCC7120; Oscillatoria sp. PCC 6506; Pelotomaculum_thermopropionicum_SI;Petrotoga mobilis SJ95; Polaromonas naphthalenivorans CJ2; Polaromonassp. JS666; Pseudoalteromonas haloplanktis TAC125; Streptomycespristinaespiralis ATCC 25486; Streptomyces pristinaespiralis ATCC 25486;Streptococcus thermophilus; Streptomyces viridochromogenes DSM 40736;Streptosporangium roseum DSM 43021; Synechococcus sp. PCC 7335; andThermosipho africanus TCF52B (Chylinski et al., RNA Biol., 2013; 10(5):726-737.

In addition to Cas9 orthologs, other Cas9 variants such as fusionproteins of inactive dCas9 and effector domains with different functionscan be served as a platform for genetic modulation. Any of the foregoingenzymes can be useful in the present disclosure.

Further examples of endonucleases that can be utilized in the presentdisclosure are provided in SEQ ID NOs: 1-612. These proteins can bemodified before use or can be encoded in a nucleic acid sequence such asa DNA, RNA or mRNA or within a vector construct such as the plasmids oradeno-associated virus (AAV) vectors taught herein. Further, they can becodon optimized.

Genome-Targeting Nucleic Acid

The present disclosure provides a genome-targeting nucleic acid that candirect the activities of an associated polypeptide (e.g., asite-directed polypeptide) to a specific target sequence within a targetnucleic acid. The genome-targeting nucleic acid can be an RNA. Agenome-targeting RNA is referred to as a “guide RNA” or “gRNA” herein. Aguide RNA can comprise at least a spacer sequence that hybridizes to atarget nucleic acid sequence of interest, and a CRISPR repeat sequence.In Type II systems, the gRNA also comprises a second RNA called thetracrRNA sequence. In the Type II gRNA, the CRISPR repeat sequence andtracrRNA sequence hybridize to each other to form a duplex. In the TypeV gRNA, the crRNA forms a duplex. In both systems, the duplex can bind asite-directed polypeptide, such that the guide RNA and site-directpolypeptide form a complex. The genome-targeting nucleic acid canprovide target specificity to the complex by virtue of its associationwith the site-directed polypeptide. The genome-targeting nucleic acidthus can direct the activity of the site-directed polypeptide.

Exemplary guide RNAs include the spacer sequences in SEQ ID NOs:5272-5313, 5398-5409, and 5434-5443 of the Sequence Listing, shown withgenome location of their target sequence (See SEQ ID NOs: 5314-5355 inFIG. 2B; 5410-5421 in FIG. 2E; and 5444-5453 in FIG. 2E) and theassociated Cas9 cut site, wherein the genome location is based on theGRCh38/hg38 human genome assembly.

Each guide RNA can be designed to include a spacer sequencecomplementary to its genomic target sequence. For example, each of thespacer sequences in SEQ ID NOs: 5272-5313, 5398-5409, and 5434-5443 ofthe Sequence Listing can be put into a single RNA chimera or a crRNA(along with a corresponding tracrRNA). See Jinek et al., Science, 337,816-821 (2012) and Deltcheva et al., Nature, 471, 602-607 (2011).

The genome-targeting nucleic acid can be a double-molecule guide RNA.The genome-targeting nucleic acid can be a single-molecule guide RNA.The double-molecule guide RNA or single-molecule guide RNA can bemodified.

A double-molecule guide RNA can comprise two strands of RNA. The firststrand comprises in the 5′ to 3′ direction, an optional spacer extensionsequence, a spacer sequence and a minimum CRISPR repeat sequence. Thesecond strand can comprise a minimum tracrRNA sequence (complementary tothe minimum CRISPR repeat sequence), a 3′ tracrRNA sequence and anoptional tracrRNA extension sequence.

A single-molecule guide RNA (sgRNA) in a Type II system can comprise, inthe 5′ to 3′ direction, an optional spacer extension sequence, a spacersequence, a minimum CRISPR repeat sequence, a single-molecule guidelinker, a minimum tracrRNA sequence, a 3′ tracrRNA sequence and anoptional tracrRNA extension sequence. The optional tracrRNA extensioncan comprise elements that contribute additional functionality (e.g.,stability) to the guide RNA. The single-molecule guide linker can linkthe minimum CRISPR repeat and the minimum tracrRNA sequence to form ahairpin structure. The optional tracrRNA extension can comprise one ormore hairpins.

The sgRNA can comprise a variable length spacer sequence with 17-30nucleotides at the 5′ end of the sgRNA sequence (Table 4). In otherexamples, the sgRNA can comprise a variable length spacer sequence with17-24 nucleotides at the 5′ end of the sgRNA sequence.

The sgRNA can comprise a 20 nucleotide spacer sequence at the 5′ end ofthe sgRNA sequence. The sgRNA can comprise a less than 20 nucleotidespacer sequence at the 5′ end of the sgRNA sequence. The sgRNA cancomprise a 19 nucleotide spacer sequence at the 5′ end of the sgRNAsequence. The sgRNA can comprise a 18 nucleotide spacer sequence at the5′ end of the sgRNA sequence. The sgRNA can comprise a 17 nucleotidespacer sequence at the 5′ end of the sgRNA sequence. The sgRNA cancomprise a more than 20 nucleotide spacer sequence at the 5′ end of thesgRNA sequence. The sgRNA can comprise a 21 nucleotide spacer sequenceat the 5′ end of the sgRNA sequence. The sgRNA can comprise a 22nucleotide spacer sequence at the 5′ end of the sgRNA sequence. ThesgRNA can comprise a 23 nucleotide spacer sequence at the 5′ end of thesgRNA sequence. The sgRNA can comprise a 24 nucleotide spacer sequenceat the 5′ end of the sgRNA sequence. The sgRNA can comprise a 25nucleotide spacer sequence at the 5′ end of the sgRNA sequence. ThesgRNA can comprise a 26 nucleotide spacer sequence at the 5′ end of thesgRNA sequence. The sgRNA can comprise a 27 nucleotide spacer sequenceat the 5′ end of the sgRNA sequence. The sgRNA can comprise a 28nucleotide spacer sequence at the 5′ end of the sgRNA sequence. ThesgRNA can comprise a 29 nucleotide spacer sequence at the 5′ end of thesgRNA sequence. The sgRNA can comprise a 30 nucleotide spacer sequenceat the 5′ end of the sgRNA sequence.

The sgRNA can comprise no uracil at the 3′end of the sgRNA sequence,such as in SEQ ID NOs: 5268, 5495, 5498, 5501, and 5504 of Table 4. ThesgRNA can comprise one or more uracil at the 3′end of the sgRNAsequence, such as in SEQ ID NO: 5269, 5267, 5494, 5496, 5497, 5499,5500, 5502, 5503, and 5505 in Table 4. For example, the sgRNA cancomprise 1 uracil (U) at the 3′ end of the sgRNA sequence. The sgRNA cancomprise 2 uracil (UU) at the 3′ end of the sgRNA sequence. The sgRNAcan comprise 3 uracil (UUU) at the 3′ end of the sgRNA sequence. ThesgRNA can comprise 4 uracil (UUUU) at the 3′ end of the sgRNA sequence.The sgRNA can comprise 5 uracil (UUUUU) at the 3′ end of the sgRNAsequence. The sgRNA can comprise 6 uracil (UUUUUU) at the 3′ end of thesgRNA sequence. The sgRNA can comprise 7 uracil (UUUUUUU) at the 3′ endof the sgRNA sequence. The sgRNA can comprise 8 uracil (UUUUUUUU) at the3′ end of the sgRNA sequence.

The sgRNA can be unmodified or modified. For example, modified sgRNAscan comprise one or more 2′-O-methyl phosphorothioate nucleotides.

TABLE 4 SEQ ID NO. sgRNA sequence 5267n₍₁₇₋₃₀₎guuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcSp accgagucggugcuuuu 5268n₍₁₇₋₃₀₎guuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaaguggcSp accgagucggugc 5269n₍₁₇₋₃₀₎guuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuaucaacuugaaaaagu Spggcaccgagucggugcu₍₁₋₈₎ 5494n₍₁₇₋₃₀₎guuuaaguacucugugcuggaaacagcacagaaucuacuuaaacaaggcaaaaugccguguuuaSa ucucgucaacuuguuggcgagauuuuuu 5495n₍₁₇₋₃₀₎guuuaaguacucugugcuggaaacagcacagaaucuacuuaaacaaggcaaaaugccguguuuaSa ucucgucaacuuguuggcgaga 5496n₍₁₇₋₃₀₎guuuaaguacucugugcuggaaacagcacagaaucuacuuaaacaaggcaaaaugccguguuuaSa ucucgucaacuuguuggcgagau₍₁₋₈₎ 5497n₍₁₇₋₃₀₎guuuuaguacucuguaaugaaaauuacagaaucuacuaaaacaaggcaaaaugccguguuuaucucSa gucaacuuguuggcgagauuuuuuuu 5498n₍₁₇₋₃₀₎guuuuaguacucuguaaugaaaauuacagaaucuacuaaaacaaggcaaaaugccguguuuaucucSa gucaacuuguuggcgaga 5499n₍₁₇₋₃₀₎guuuuaguacucuguaaugaaaauuacagaaucuacuaaaacaaggcaaaaugccguguuuaucucSa gucaacuuguuggcgagau₍₁₋₈₎ 5500n₍₁₇₋₃₀₎guuuaaguacucugugcuggaaacagcacagaaucuacuuaaacaaggcaaaaugccguguuuaucSa ucgucaacuuguuggcgagauuuuuuuu 5501n₍₁₇₋₃₀₎guuuaaguacucugugcuggaaacagcacagaaucuacuuaaacaaggcaaaaugccguguuuaucSa ucgucaacuuguuggcgaga 5502n₍₁₇₋₃₀₎guuuaaguacucugugcuggaaacagcacagaaucuacuuaaacaaggcaaaaugccguguuuaucSa ucgucaacuuguuggcgagau₍₁₋₈₎ 5503n₍₁₇₋₃₀₎guuuuaguacucuggaaacagaaucuacuaaaacaaggcaaaaugccguguuuaucucgucaacuuSa guuggcgagauuuu 5504n₍₁₇₋₃₀₎guuuuaguacucuggaaacagaaucuacuaaaacaaggcaaaaugccguguuuaucucgucaacuuSa guuggcgaga 5505n₍₁₇₋₃₀₎guuuuaguacucuggaaacagaaucuacuaaaacaaggcaaaaugccguguuuaucucgucaacuuSa guuggcgagau₍₁₋₈₎

A single-molecule guide RNA (sgRNA) in a Type V system can comprise, inthe 5′ to 3′ direction, a minimum CRISPR repeat sequence and a spacersequence.

By way of illustration, guide RNAs used in the CRISPR/Cas/Cpf1 system,or other smaller RNAs, can be readily synthesized by chemical means, asillustrated below and described in the art. While chemical syntheticprocedures are continually expanding, purifications of such RNAs byprocedures such as high performance liquid chromatography (HPLC, whichavoids the use of gels such as PAGE) tends to become more challenging aspolynucleotide lengths increase significantly beyond a hundred or sonucleotides. One approach used for generating RNAs of greater length isto produce two or more molecules that are ligated together. Much longerRNAs, such as those encoding a Cas9 or Cpf1 endonuclease, are morereadily generated enzymatically. Various types of RNA modifications canbe introduced during or after chemical synthesis and/or enzymaticgeneration of RNAs, e.g., modifications that enhance stability, reducethe likelihood or degree of innate immune response, and/or enhance otherattributes, as described in the art.

gRNAs or sgRNAs that Target the R838H Mutation in a GUCY2D Gene

The present disclosure provides one or more gRNAs (or sgRNAs) forediting a R838H mutation in a GUCY2D gene in a cell from a patient withautosomal dominant CORD (FIGS. 2A and 2D).

The one or more gRNAs can comprise a spacer sequence selected from thegroup consisting of nucleic acid sequences in SEQ ID NOs: 5282-5293 ofthe Sequence Listing. These gRNA sequences have zero mismatches with theR838H GUCY2D allele and 1 mismatch with the wild-type GUCY2D allele.

The one or more gRNAs can comprise a spacer sequence selected from thegroup consisting of nucleic acid sequences in SEQ ID NOs: 5398-5409 ofthe Sequence Listing. These gRNA sequences have 1 mismatch with theR838H GUCY2D allele and 2 mismatches with the wild-type GUCY2D allele.

gRNAs or sgRNAs disclosed herein can associate with a DNA endonucleaseto form a ribonucleoprotein complex, which stably edits the R838Hmutation in a GUCY2D gene. This editing is not transient.

gRNAs or sgRNAs that Target the R838C Mutation in a GUCY2D Gene

The present disclosure provides one or more gRNAs (or sgRNAs) forediting a R838C mutation in a GUCY2D gene in a cell from a patient withautosomal dominant CORD (FIGS. 2A and 2D).

The one or more gRNAs can comprise a spacer sequence selected from thegroup consisting of nucleic acid sequences in SEQ ID NOs: 5294-5303 ofthe Sequence Listing. These gRNA sequences have zero mismatches with theR838C GUCY2D allele and 1 mismatch with the wild-type GUCY2D allele.

The one or more gRNAs can comprise a spacer sequence selected from thegroup consisting of nucleic acid sequences in 5398-5409 of the SequenceListing. These gRNA sequences have 1 mismatch with the R838C GUCY2Dallele and 2 mismatches with the wild-type GUCY2D allele.

gRNAs or sgRNAs disclosed herein can associate with a DNA endonucleaseto form a ribonucleoprotein complex, which stably edits the R838Cmutation in a GUCY2D gene. This editing is not transient.

gRNAs or sgRNAs that Target the R838S Mutation in a GUCY2D Gene

The present disclosure provides one or more gRNAs (or sgRNAs) forediting a R838S mutation in a GUCY2D gene in a cell from a patient withautosomal dominant CORD (FIGS. 2A and 2D).

The one or more gRNAs can comprise a spacer sequence selected from thegroup consisting of nucleic acid sequences in SEQ ID NOs: 5304-5313 ofthe Sequence Listing. These gRNA sequences have zero mismatches with theR838S GUCY2D allele and 1 mismatch with the wild-type GUCY2D allele.

The one or more gRNAs can comprise a spacer sequence selected from thegroup consisting of nucleic acid sequences in 5434-5443 of the SequenceListing. These gRNA sequences have 1 mismatch with the R838S GUCY2Dallele and 2 mismatches with the wild-type GUCY2D allele.

gRNAs or sgRNAs disclosed herein can associate with a DNA endonucleaseto form a ribonucleoprotein complex, which stably edits the R838Smutation in a GUCY2D gene. This editing is not transient.

“Double Mutation” Guide RNAs

The present disclosure provides gRNAs (or sgRNAs), referred to hereinas, “double mutation” guide RNAs, that can edit multiple mutant allelesof the GUCY2D gene.

In certain examples, the double mutation guide RNAs can comprise aspacer sequence comprising 1 mismatched base with, for example, an R838Cmutant allele and comprising a second mismatched base with, for example,a R838H mutant allele. Double mutation gRNAs that target the R838C andR838H mutant alleles are referred to herein as “R838CH double mutation”gRNAs.

In certain other examples, the double mutation guide RNAs can comprise aspacer sequence comprising 1 mismatched base with, for example, an R838Smutant allele and comprising a second mismatched based with, for examplea R838H mutant allele. Double mutation gRNAs that target the R838S andR838H mutant alleles are referred to herein as “R838SH double mutation”gRNAs.

The double mutation guide RNAs (e.g., R838CH double mutation gRNAs orR838SH double mutation gRNAs) can comprise 2 mismatched bases with awild-type allele. The double mutation guide RNAs can comprise reducedoff-target editing of a wild-type GUCY2D allele.

gRNAs or sgRNAs that Target the R838C and R838H Mutations in a GUCY2DGene (R838CH Double Mutation gRNAs)

The present disclosure provides gRNAs (or sgRNAs) for editing a R838C orR838H mutation in a GUCY2D gene in a cell from a patient with autosomaldominant CORD (FIG. 2D).

The gRNAs can comprise a spacer sequence selected from the groupconsisting of nucleic acid sequences in SEQ ID NOs: 5398-5409 of theSequence Listing. These gRNA sequences have 1 mismatch with the R838CGUCY2D allele and R838H GUCY2D allele. These gRNA sequences have 2mismatches with the wild-type GUCY2D allele.

These gRNAs can be used to treat patients with a R838H mutation in theGUCY2D gene or patients with an R838C mutation in the GUCY2D gene. ThesegRNAs can be specific for both the R838H and R838C mutant allele becausethey have two consecutive mismatches compared to a sequence thatcorresponds with the wild-type codon 838. The two consecutive mismatchesin the gRNA sequence reduce the probability that the wild-type GUCY2Dsequence will be cleaved because the two mismatches within the gRNA areconsecutive and not separated (FIG. 3 of Klein et al “HybridizationKinetics Explains CRISPR-Cas Off-Targeting Rules”, Cell Reports 22,February 2018, pages 1413-1423).

gRNAs or sgRNAs disclosed herein can associate with a DNA endonucleaseto form a ribonucleoprotein complex, which stably edits either the R838Cmutation or R838H mutation in a GUCY2D gene. This editing is nottransient.

gRNAs or sgRNAs that Target the R838S and R838H Mutations in a GUCY2DGene (R838SH Double Mutation gRNAs)

The present disclosure provides gRNAs (or sgRNAs) for editing a R838S orR838H mutation in a GUCY2D gene in a cell from a patient with autosomaldominant CORD (FIG. 2D).

The gRNAs can comprise a spacer sequence selected from the groupconsisting of nucleic acid sequences in SEQ ID NOs: 5434-5443 of theSequence Listing. These gRNA sequences have 1 mismatch with the R838SGUCY2D allele and R838H GUCY2D allele. These gRNA sequences have 2mismatches with the wild-type GUCY2D allele.

These gRNAs can be used to treat patients with a R838S mutation in theGUCY2D gene or patients with an R838H mutation in the GUCY2D gene. ThesegRNAs can be specific for both the R838S and R838H mutant allele becausethey have two consecutive mismatches compared to a sequence thatcorresponds with the wild-type codon 838. As described above, the twoconsecutive mismatches reduce the probability that the wild-type GUCY2Dsequence will be cleaved because the two mismatches within the gRNA areconsecutive and not separated.

gRNAs or sgRNAs disclosed herein can associate with a DNA endonucleaseto form a ribonucleoprotein complex, which stably edits either the R838Smutation or R838H mutation in a GUCY2D gene. This editing is nottransient.

Spacer Extension Sequence

In some examples of genome-targeting nucleic acids, a spacer extensionsequence can modify activity, provide stability and/or provide alocation for modifications of a genome-targeting nucleic acid. A spacerextension sequence can modify on- or off-target activity or specificity.In some examples, a spacer extension sequence can be provided. Thespacer extension sequence can have a length of more than 1, 5, 10, 15,20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180,200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 1000, 2000, 3000,4000, 5000, 6000, or 7000 or more nucleotides. The spacer extensionsequence can have a length of less than 1, 5, 10, 15, 20, 25, 30, 35,40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260,280, 300, 320, 340, 360, 380, 400, 1000, 2000, 3000, 4000, 5000, 6000,7000 or more nucleotides. The spacer extension sequence can be less than10 nucleotides in length. The spacer extension sequence can be between10-30 nucleotides in length. The spacer extension sequence can bebetween 30-70 nucleotides in length.

The spacer extension sequence can comprise another moiety (e.g., astability control sequence, an endoribonuclease binding sequence, aribozyme). The moiety can decrease or increase the stability of anucleic acid targeting nucleic acid. The moiety can be a transcriptionalterminator segment (i.e., a transcription termination sequence). Themoiety can function in a eukaryotic cell. The moiety can function in aprokaryotic cell. The moiety can function in both eukaryotic andprokaryotic cells. Non-limiting examples of suitable moieties include: a5′ cap (e.g., a 7-methylguanylate cap (m7 G)), a riboswitch sequence(e.g., to allow for regulated stability and/or regulated accessibilityby proteins and protein complexes), a sequence that forms a dsRNA duplex(i.e., a hairpin), a sequence that targets the RNA to a subcellularlocation (e.g., nucleus, mitochondria, chloroplasts, and the like), amodification or sequence that provides for tracking (e.g., directconjugation to a fluorescent molecule, conjugation to a moiety thatfacilitates fluorescent detection, a sequence that allows forfluorescent detection, etc.), and/or a modification or sequence thatprovides a binding site for proteins (e.g., proteins that act on DNA,including transcriptional activators, transcriptional repressors, DNAmethyltransferases, DNA demethylases, histone acetyltransferases,histone deacetylases, and the like).

Spacer Sequence

The spacer sequence hybridizes to a sequence in a target nucleic acid ofinterest. The spacer of a genome-targeting nucleic acid can interactwith a target nucleic acid in a sequence-specific manner viahybridization (i.e., base pairing). The nucleotide sequence of thespacer can vary depending on the sequence of the target nucleic acid ofinterest. In a CRISPR/Cas system herein, the spacer sequence can bedesigned to hybridize to a target nucleic acid that is located 5′ of aPAM of the Cas9 or Cpf1 enzyme used in the system. The spacer canperfectly match the target sequence or can have mismatches. Each Cas9enzyme has a particular PAM sequence that it recognizes in a target DNA.For example, S. pyogenes recognizes in a target nucleic acid a PAM thatcomprises the sequence 5′-NRG-3′, where R comprises either A or G, whereN is any nucleotide and N is immediately 3′ of the target nucleic acidsequence targeted by the spacer sequence. For example, S. aureus Cas9recognizes in a target nucleic acid a PAM that comprises the sequence5′-NNGRRT-3′, where R comprises either A or G, where N is any nucleotideand N is immediately 3′ of the target nucleic acid sequence targeted bythe spacer sequence. In certain examples, S. aureus Cas9 recognizes in atarget nucleic acid a PAM that comprises the sequence 5′-NNGRRN-3′,where R comprises either A or G, where N is any nucleotide and the N isimmediately 3′ of the target nucleic acid sequence targeted by thespacer sequence. For example, C. jejuni recognizes in a target nucleicacid a PAM that comprises the sequence 5′-NNNNACA-3′ or 5′-NNNNACAC-3′,where N is any nucleotide and N is immediately 3′ of the target nucleicacid sequence targeted by the spacer sequence. In certain examples, C.jejuni Cas9 recognizes in a target nucleic acid a PAM that comprises thesequence 5′-NNNVRYM-3′ or 5′-NNVRYAC-3′, where V comprises either A, Gor C, where R comprises either A or G, where Y comprises either C or T,where M comprises A or C, where N is any nucleotide and the N isimmediately 3′ of the target nucleic acid sequence targeted by thespacer sequence.

The target nucleic acid sequence can comprise 20 nucleotides. The targetnucleic acid can comprise less than 20 nucleotides. The target nucleicacid can comprise more than 20 nucleotides. The target nucleic acid cancomprise at least: 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30or more nucleotides. The target nucleic acid can comprise at most: 5,10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides.The target nucleic acid sequence can comprise 20 bases immediately 5′ ofthe first nucleotide of the PAM. For example, in a sequence comprising5′-NNNNNNNNNNNNNNNNNNNNNRG-3′ (SEQ ID NO. 5270), the target nucleic acidcan comprise the sequence that corresponds to the Ns, wherein N is anynucleotide, and the underlined NRG sequence is the S. pyogenes PAM.

The spacer sequence that hybridizes to the target nucleic acid can havea length of at least about 6 nucleotides (nt). The spacer sequence canbe at least about 6 nt, at least about 10 nt, at least about 15 nt, atleast about 18 nt, at least about 19 nt, at least about 20 nt, at leastabout 25 nt, at least about 30 nt, at least about 35 nt or at leastabout 40 nt, from about 6 nt to about 80 nt, from about 6 nt to about 50nt, from about 6 nt to about 45 nt, from about 6 nt to about 40 nt, fromabout 6 nt to about 35 nt, from about 6 nt to about 30 nt, from about 6nt to about 25 nt, from about 6 nt to about 20 nt, from about 6 nt toabout 19 nt, from about 10 nt to about 50 nt, from about 10 nt to about45 nt, from about 10 nt to about 40 nt, from about 10 nt to about 35 nt,from about 10 nt to about 30 nt, from about 10 nt to about 25 nt, fromabout 10 nt to about 20 nt, from about 10 nt to about 19 nt, from about19 nt to about 25 nt, from about 19 nt to about 30 nt, from about 19 ntto about 35 nt, from about 19 nt to about 40 nt, from about 19 nt toabout 45 nt, from about 19 nt to about 50 nt, from about 19 nt to about60 nt, from about 20 nt to about 25 nt, from about 20 nt to about 30 nt,from about 20 nt to about 35 nt, from about 20 nt to about 40 nt, fromabout 20 nt to about 45 nt, from about 20 nt to about 50 nt, or fromabout 20 nt to about 60 nt. In some examples, the spacer can comprise 24nucleotides. In some examples, the spacer can comprise 23 nucleotides.In some examples, the spacer can comprise 22 nucleotides. In someexamples, the spacer can comprise 21 nucleotides. In some examples, thespacer sequence can comprise 20 nucleotides. In some examples, thespacer can comprise 19 nucleotides. In some examples, the spacer cancomprise 18 nucleotides. In some examples, the spacer can comprise 17nucleotides.

In some examples, the percent complementarity between the spacersequence and the target nucleic acid is at least about 30%, at leastabout 40%, at least about 50%, at least about 60%, at least about 65%,at least about 70%, at least about 75%, at least about 80%, at leastabout 85%, at least about 90%, at least about 95%, at least about 97%,at least about 98%, at least about 99%, or 100%. In some examples, thepercent complementarity between the spacer sequence and the targetnucleic acid is at most about 30%, at most about 40%, at most about 50%,at most about 60%, at most about 65%, at most about 70%, at most about75%, at most about 80%, at most about 85%, at most about 90%, at mostabout 95%, at most about 97%, at most about 98%, at most about 99%, or100%. In some examples, the percent complementarity between the spacersequence and the target nucleic acid is 100% over the six contiguous5′-most nucleotides of the target sequence of the complementary strandof the target nucleic acid. The percent complementarity between thespacer sequence and the target nucleic acid can be at least 60% overabout 20 contiguous nucleotides. The length of the spacer sequence andthe target nucleic acid can differ by 1 to 6 nucleotides, which can bethought of as a bulge or bulges.

The spacer sequence can be designed or chosen using a computer program.The computer program can use variables, such as predicted meltingtemperature, secondary structure formation, predicted annealingtemperature, sequence identity, genomic context, chromatinaccessibility, % GC, frequency of genomic occurrence (e.g., of sequencesthat are identical or are similar but vary in one or more spots as aresult of mismatch, insertion or deletion), methylation status, presenceof SNPs, and the like.

Minimum CRISPR Repeat Sequence

A minimum CRISPR repeat sequence can be a sequence with at least about30%, about 40%, about 50%, about 60%, about 65%, about 70%, about 75%,about 80%, about 85%, about 90%, about 95%, or 100% sequence identity toa reference CRISPR repeat sequence (e.g., crRNA from S. pyogenes).

A minimum CRISPR repeat sequence can comprise nucleotides that canhybridize to a minimum tracrRNA sequence in a cell. The minimum CRISPRrepeat sequence and a minimum tracrRNA sequence can form a duplex, i.e.a base-paired double-stranded structure. Together, the minimum CRISPRrepeat sequence and the minimum tracrRNA sequence can bind to thesite-directed polypeptide. At least a part of the minimum CRISPR repeatsequence can hybridize to the minimum tracrRNA sequence. At least a partof the minimum CRISPR repeat sequence can comprise at least about 30%,about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about80%, about 85%, about 90%, about 95%, or 100% complementary to theminimum tracrRNA sequence. At least a part of the minimum CRISPR repeatsequence can comprise at most about 30%, about 40%, about 50%, about60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%,about 95%, or 100% complementary to the minimum tracrRNA sequence.

The minimum CRISPR repeat sequence can have a length from about 7nucleotides to about 100 nucleotides. For example, the length of theminimum CRISPR repeat sequence is from about 7 nucleotides (nt) to about50 nt, from about 7 nt to about 40 nt, from about 7 nt to about 30 nt,from about 7 nt to about 25 nt, from about 7 nt to about 20 nt, fromabout 7 nt to about 15 nt, from about 8 nt to about 40 nt, from about 8nt to about 30 nt, from about 8 nt to about 25 nt, from about 8 nt toabout 20 nt, from about 8 nt to about 15 nt, from about 15 nt to about100 nt, from about 15 nt to about 80 nt, from about 15 nt to about 50nt, from about 15 nt to about 40 nt, from about 15 nt to about 30 nt, orfrom about 15 nt to about 25 nt. In some examples, the minimum CRISPRrepeat sequence can be approximately 9 nucleotides in length. Theminimum CRISPR repeat sequence can be approximately 12 nucleotides inlength.

The minimum CRISPR repeat sequence can be at least about 60% identicalto a reference minimum CRISPR repeat sequence (e.g., wild-type crRNAfrom S. pyogenes) over a stretch of at least 6, 7, or 8 contiguousnucleotides. For example, the minimum CRISPR repeat sequence can be atleast about 65% identical, at least about 70% identical, at least about75% identical, at least about 80% identical, at least about 85%identical, at least about 90% identical, at least about 95% identical,at least about 98% identical, at least about 99% identical or 100%identical to a reference minimum CRISPR repeat sequence over a stretchof at least 6, 7, or 8 contiguous nucleotides.

Minimum tracrRNA Sequence

A minimum tracrRNA sequence can be a sequence with at least about 30%,about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about80%, about 85%, about 90%, about 95%, or 100% sequence identity to areference tracrRNA sequence (e.g., wild type tracrRNA from S. pyogenes).

A minimum tracrRNA sequence can comprise nucleotides that hybridize to aminimum CRISPR repeat sequence in a cell. A minimum tracrRNA sequenceand a minimum CRISPR repeat sequence form a duplex, i.e. a base-paireddouble-stranded structure. Together, the minimum tracrRNA sequence andthe minimum CRISPR repeat can bind to a site-directed polypeptide. Atleast a part of the minimum tracrRNA sequence can hybridize to theminimum CRISPR repeat sequence. The minimum tracrRNA sequence can be atleast about 30%, about 40%, about 50%, about 60%, about 65%, about 70%,about 75%, about 80%, about 85%, about 90%, about 95%, or 100%complementary to the minimum CRISPR repeat sequence.

The minimum tracrRNA sequence can have a length from about 7 nucleotidesto about 100 nucleotides. For example, the minimum tracrRNA sequence canbe from about 7 nucleotides (nt) to about 50 nt, from about 7 nt toabout 40 nt, from about 7 nt to about 30 nt, from about 7 nt to about 25nt, from about 7 nt to about 20 nt, from about 7 nt to about 15 nt, fromabout 8 nt to about 40 nt, from about 8 nt to about 30 nt, from about 8nt to about 25 nt, from about 8 nt to about 20 nt, from about 8 nt toabout 15 nt, from about 15 nt to about 100 nt, from about 15 nt to about80 nt, from about 15 nt to about 50 nt, from about 15 nt to about 40 nt,from about 15 nt to about 30 nt or from about 15 nt to about 25 nt long.The minimum tracrRNA sequence can be approximately 9 nucleotides inlength. The minimum tracrRNA sequence can be approximately 12nucleotides. The minimum tracrRNA can consist of tracrRNA nt 23-48described in Jinek et al., supra.

The minimum tracrRNA sequence can be at least about 60% identical to areference minimum tracrRNA (e.g., wild type, tracrRNA from S. pyogenes)sequence over a stretch of at least 6, 7, or 8 contiguous nucleotides.For example, the minimum tracrRNA sequence can be at least about 65%identical, about 70% identical, about 75% identical, about 80%identical, about 85% identical, about 90% identical, about 95%identical, about 98% identical, about 99% identical or 100% identical toa reference minimum tracrRNA sequence over a stretch of at least 6, 7,or 8 contiguous nucleotides.

The duplex between the minimum CRISPR RNA and the minimum tracrRNA cancomprise a double helix. The duplex between the minimum CRISPR RNA andthe minimum tracrRNA can comprise at least about 1, 2, 3, 4, 5, 6, 7, 8,9, or 10 or more nucleotides. The duplex between the minimum CRISPR RNAand the minimum tracrRNA can comprise at most about 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 or more nucleotides.

The duplex can comprise a mismatch (i.e., the two strands of the duplexare not 100% complementary). The duplex can comprise at least about 1,2, 3, 4, or 5 or mismatches. The duplex can comprise at most about 1, 2,3, 4, or 5 or mismatches. The duplex can comprise no more than 2mismatches.

Bulges

In some cases, there can be a “bulge” in the duplex between the minimumCRISPR RNA and the minimum tracrRNA. A bulge is an unpaired region ofnucleotides within the duplex. A bulge can contribute to the binding ofthe duplex to the site-directed polypeptide. The bulge can comprise, onone side of the duplex, an unpaired 5′-XXXY-3′ where X is any purine andY comprises a nucleotide that can form a wobble pair with a nucleotideon the opposite strand, and an unpaired nucleotide region on the otherside of the duplex. The number of unpaired nucleotides on the two sidesof the duplex can be different.

In one example, the bulge can comprise an unpaired purine (e.g.,adenine) on the minimum CRISPR repeat strand of the bulge. In someexamples, the bulge can comprise an unpaired 5′-AAGY-3′ of the minimumtracrRNA sequence strand of the bulge, where Y comprises a nucleotidethat can form a wobble pairing with a nucleotide on the minimum CRISPRrepeat strand.

A bulge on the minimum CRISPR repeat side of the duplex can comprise atleast 1, 2, 3, 4, or 5 or more unpaired nucleotides. A bulge on theminimum CRISPR repeat side of the duplex can comprise at most 1, 2, 3,4, or 5 or more unpaired nucleotides. A bulge on the minimum CRISPRrepeat side of the duplex can comprise 1 unpaired nucleotide.

A bulge on the minimum tracrRNA sequence side of the duplex can compriseat least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more unpaired nucleotides.A bulge on the minimum tracrRNA sequence side of the duplex can compriseat most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more unpaired nucleotides. Abulge on a second side of the duplex (e.g., the minimum tracrRNAsequence side of the duplex) can comprise 4 unpaired nucleotides.

A bulge can comprise at least one wobble pairing. In some examples, abulge can comprise at most one wobble pairing. A bulge can comprise atleast one purine nucleotide. A bulge can comprise at least 3 purinenucleotides. A bulge sequence can comprise at least 5 purinenucleotides. A bulge sequence can comprise at least one guaninenucleotide. In some examples, a bulge sequence can comprise at least oneadenine nucleotide.

Hairpins

In various examples, one or more hairpins can be located 3′ to theminimum tracrRNA in the 3′ tracrRNA sequence.

The hairpin can start at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,or 20 or more nucleotides 3′ from the last paired nucleotide in theminimum CRISPR repeat and minimum tracrRNA sequence duplex. The hairpincan start at most about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or morenucleotides 3′ of the last paired nucleotide in the minimum CRISPRrepeat and minimum tracrRNA sequence duplex.

The hairpin can comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,15, or 20 or more consecutive nucleotides. The hairpin can comprise atmost about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or more consecutivenucleotides.

The hairpin can comprise a CC dinucleotide (i.e., two consecutivecytosine nucleotides).

The hairpin can comprise duplexed nucleotides (e.g., nucleotides in ahairpin, hybridized together). For example, a hairpin can comprise a CCdinucleotide that is hybridized to a GG dinucleotide in a hairpin duplexof the 3′ tracrRNA sequence.

One or more of the hairpins can interact with guide RNA-interactingregions of a site-directed polypeptide.

In some examples, there are two or more hairpins, and in other examplesthere are three or more hairpins.

3′ tracrRNA Sequence

A 3′ tracrRNA sequence can comprise a sequence with at least about 30%,about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, about80%, about 85%, about 90%, about 95%, or 100% sequence identity to areference tracrRNA sequence (e.g., a tracrRNA from S. pyogenes).

The 3′ tracrRNA sequence can have a length from about 6 nucleotides toabout 100 nucleotides. For example, the 3′ tracrRNA sequence can have alength from about 6 nucleotides (nt) to about 50 nt, from about 6 nt toabout 40 nt, from about 6 nt to about 30 nt, from about 6 nt to about 25nt, from about 6 nt to about 20 nt, from about 6 nt to about 15 nt, fromabout 8 nt to about 40 nt, from about 8 nt to about 30 nt, from about 8nt to about 25 nt, from about 8 nt to about 20 nt, from about 8 nt toabout 15 nt, from about 15 nt to about 100 nt, from about 15 nt to about80 nt, from about 15 nt to about 50 nt, from about 15 nt to about 40 nt,from about 15 nt to about 30 nt, or from about 15 nt to about 25 nt. The3′ tracrRNA sequence can have a length of approximately 14 nucleotides.

The 3′ tracrRNA sequence can be at least about 60% identical to areference 3′ tracrRNA sequence (e.g., wild type 3′ tracrRNA sequencefrom S. pyogenes) over a stretch of at least 6, 7, or 8 contiguousnucleotides. For example, the 3′ tracrRNA sequence can be at least about60% identical, about 65% identical, about 70% identical, about 75%identical, about 80% identical, about 85% identical, about 90%identical, about 95% identical, about 98% identical, about 99%identical, or 100% identical, to a reference 3′ tracrRNA sequence (e.g.,wild type 3′ tracrRNA sequence from S. pyogenes) over a stretch of atleast 6, 7, or 8 contiguous nucleotides.

The 3′ tracrRNA sequence can comprise more than one duplexed region(e.g., hairpin, hybridized region). The 3′ tracrRNA sequence cancomprise two duplexed regions.

The 3′ tracrRNA sequence can comprise a stem loop structure. The stemloop structure in the 3′ tracrRNA can comprise at least 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 15 or 20 or more nucleotides. The stem loop structure inthe 3′ tracrRNA can comprise at most 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 ormore nucleotides. The stem loop structure can comprise a functionalmoiety. For example, the stem loop structure can comprise an aptamer, aribozyme, a protein-interacting hairpin, a CRISPR array, an intron, oran exon. The stem loop structure can comprise at least about 1, 2, 3, 4,or 5 or more functional moieties. The stem loop structure can compriseat most about 1, 2, 3, 4, or 5 or more functional moieties.

The hairpin in the 3′ tracrRNA sequence can comprise a P-domain. In someexamples, the P-domain can comprise a double-stranded region in thehairpin.

tracrRNA Extension Sequence

A tracrRNA extension sequence can be provided whether the tracrRNA is inthe context of single-molecule guides or double-molecule guides. ThetracrRNA extension sequence can have a length from about 1 nucleotide toabout 400 nucleotides. The tracrRNA extension sequence can have a lengthof more than 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90,100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360,380, or 400 nucleotides. The tracrRNA extension sequence can have alength from about 20 to about 5000 or more nucleotides. The tracrRNAextension sequence can have a length of more than 1000 nucleotides.

The tracrRNA extension sequence can have a length of less than 1, 5, 10,15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180,200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400 or morenucleotides. The tracrRNA extension sequence can have a length of lessthan 1000 nucleotides. The tracrRNA extension sequence can comprise lessthan 10 nucleotides in length. The tracrRNA extension sequence can be10-30 nucleotides in length. The tracrRNA extension sequence can be30-70 nucleotides in length.

The tracrRNA extension sequence can comprise a functional moiety (e.g.,a stability control sequence, ribozyme, endoribonuclease bindingsequence). The functional moiety can comprise a transcriptionalterminator segment (i.e., a transcription termination sequence). Thefunctional moiety can have a total length from about 10 nucleotides (nt)to about 100 nucleotides, from about 10 nt to about 20 nt, from about 20nt to about 30 nt, from about 30 nt to about 40 nt, from about 40 nt toabout 50 nt, from about 50 nt to about 60 nt, from about 60 nt to about70 nt, from about 70 nt to about 80 nt, from about 80 nt to about 90 nt,or from about 90 nt to about 100 nt, from about 15 nt to about 80 nt,from about 15 nt to about 50 nt, from about 15 nt to about 40 nt, fromabout 15 nt to about 30 nt, or from about 15 nt to about 25 nt. Thefunctional moiety can function in a eukaryotic cell. The functionalmoiety can function in a prokaryotic cell. The functional moiety canfunction in both eukaryotic and prokaryotic cells.

Non-limiting examples of suitable tracrRNA extension functional moietiesinclude a 3′ poly-adenylated tail, a riboswitch sequence (e.g., to allowfor regulated stability and/or regulated accessibility by proteins andprotein complexes), a sequence that forms a dsRNA duplex (i.e., ahairpin), a sequence that targets the RNA to a subcellular location(e.g., nucleus, mitochondria, chloroplasts, and the like), amodification or sequence that provides for tracking (e.g., directconjugation to a fluorescent molecule, conjugation to a moiety thatfacilitates fluorescent detection, a sequence that allows forfluorescent detection, etc.), and/or a modification or sequence thatprovides a binding site for proteins (e.g., proteins that act on DNA,including transcriptional activators, transcriptional repressors, DNAmethyltransferases, DNA demethylases, histone acetyltransferases,histone deacetylases, and the like). The tracrRNA extension sequence cancomprise a primer binding site or a molecular index (e.g., barcodesequence). The tracrRNA extension sequence can comprise one or moreaffinity tags.

Single-Molecule Guide Linker Sequence

The linker sequence of a single-molecule guide nucleic acid can have alength from about 3 nucleotides to about 100 nucleotides. In Jinek etal., supra, for example, a simple 4 nucleotide “tetraloop” (-GAAA-) wasused, Science, 337(6096):816-821 (2012). An illustrative linker has alength from about 3 nucleotides (nt) to about 90 nt, from about 3 nt toabout 80 nt, from about 3 nt to about 70 nt, from about 3 nt to about 60nt, from about 3 nt to about 50 nt, from about 3 nt to about 40 nt, fromabout 3 nt to about 30 nt, from about 3 nt to about 20 nt, from about 3nt to about 10 nt. For example, the linker can have a length from about3 nt to about 5 nt, from about 5 nt to about 10 nt, from about 10 nt toabout 15 nt, from about 15 nt to about 20 nt, from about 20 nt to about25 nt, from about 25 nt to about 30 nt, from about 30 nt to about 35 nt,from about 35 nt to about 40 nt, from about 40 nt to about 50 nt, fromabout 50 nt to about 60 nt, from about 60 nt to about 70 nt, from about70 nt to about 80 nt, from about 80 nt to about 90 nt, or from about 90nt to about 100 nt. The linker of a single-molecule guide nucleic acidcan be between 4 and 40 nucleotides. The linker can be at least about100, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500,6000, 6500, or 7000 or more nucleotides. The linker can be at most about100, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500,6000, 6500, or 7000 or more nucleotides.

Linkers can comprise any of a variety of sequences, although in someexamples the linker will not comprise sequences that have extensiveregions of homology with other portions of the guide RNA, which mightcause intramolecular binding that could interfere with other functionalregions of the guide. In Jinek et al., supra, a simple 4 nucleotidesequence -GAAA- was used, Science, 337(6096):816-821 (2012), butnumerous other sequences, including longer sequences can likewise beused.

The linker sequence can comprise a functional moiety. For example, thelinker sequence can comprise one or more features, including an aptamer,a ribozyme, a protein-interacting hairpin, a protein binding site, aCRISPR array, an intron, or an exon. The linker sequence can comprise atleast about 1, 2, 3, 4, or 5 or more functional moieties. In someexamples, the linker sequence can comprise at most about 1, 2, 3, 4, or5 or more functional moieties.

Complexes of a Genome-Targeting Nucleic Acid and a Site-DirectedPolypeptide

A genome-targeting nucleic acid interacts with a site-directedpolypeptide (e.g., a nucleic acid-guided nuclease such as Cas9), therebyforming a complex. The genome-targeting nucleic acid guides thesite-directed polypeptide to a target nucleic acid.

Ribonucleoprotein Complexes (RNPs)

The site-directed polypeptide and genome-targeting nucleic acid can eachbe administered separately to a cell or a patient. On the other hand,the site-directed polypeptide can be pre-complexed with one or moreguide RNAs, or one or more crRNA together with a tracrRNA. Thesite-directed polypeptide can be pre-complexed with one or more sgRNA.The pre-complexed material can then be administered to a cell or apatient. Such pre-complexed material is known as a ribonucleoproteinparticle (RNP). The site-directed polypeptide in the RNP can be, forexample, a Cas9 endonuclease or a Cpf1 endonuclease. The site-directedpolypeptide can be flanked at the N-terminus, the C-terminus, or boththe N-terminus and C-terminus by one or more nuclear localizationsignals (NLSs). For example, a Cas9 endonuclease can be flanked by twoNLSs, one NLS located at the N-terminus and the second NLS located atthe C-terminus. The NLS can be any NLS known in the art, such as a SV40NLS. The weight ratio of genome-targeting nucleic acid to site-directedpolypeptide in the RNP can be 1:1. For example, the weight ratio ofsgRNA to Cas9 endonuclease in the RNP can be 1:1.

Target Sequence Selection

Shifts in the location of the 5′ boundary and/or the 3′ boundaryrelative to particular reference loci can be used to facilitate orenhance particular applications of gene editing, which depend in part onthe endonuclease system selected for the editing, as further describedand illustrated herein.

In a first non-limiting example of such target sequence selection, manyendonuclease systems have rules or criteria that can guide the initialselection of potential target sites for cleavage, such as therequirement of a PAM sequence motif in a particular position adjacent tothe DNA cleavage sites in the case of CRISPR Type II or Type Vendonucleases.

In another nonlimiting example of target sequence selection oroptimization, the frequency of off-target activity for a particularcombination of target sequence and gene editing endonuclease can beassessed relative to the frequency of on-target activity. In some cases,cells that have been correctly edited at the desired locus can have aselective advantage relative to other cells. Illustrative, butnonlimiting, examples of a selective advantage include the acquisitionof attributes such as enhanced rates of replication, persistence,resistance to certain conditions, enhanced rates of successfulengraftment or persistence in vivo following introduction into apatient, and other attributes associated with the maintenance orincreased numbers or viability of such cells. In other cases, cells thathave been correctly edited at the desired locus can be positivelyselected for by one or more screening methods used to identify, sort orotherwise select for cells that have been correctly edited. Bothselective advantage and directed selection methods can take advantage ofthe phenotype associated with the correction. In some cases, cells canbe edited two or more times in order to create a second modificationthat creates a new phenotype that is used to select or purify theintended population of cells. Such a second modification could becreated by adding a second gRNA for a selectable or screenable marker.In some cases, cells can be correctly edited at the desired locus usinga DNA fragment that contains the cDNA and also a selectable marker.

Whether any selective advantage is applicable or any directed selectionis to be applied in a particular case, target sequence selection canalso be guided by consideration of off-target frequencies in order toenhance the effectiveness of the application and/or reduce the potentialfor undesired alterations at sites other than the desired target. Asdescribed further and illustrated herein and in the art, the occurrenceof off-target activity can be influenced by a number of factorsincluding similarities and dissimilarities between the target site andvarious off-target sites, as well as the particular endonuclease used.Bioinformatics tools are available that assist in the prediction ofoff-target activity, and frequently such tools can also be used toidentify the most likely sites of off-target activity, which can then beassessed in experimental settings to evaluate relative frequencies ofoff-target to on-target activity, thereby allowing the selection ofsequences that have higher relative on-target activities. Illustrativeexamples of such techniques are provided herein, and others are known inthe art.

gRNAs of the present disclosure can direct editing at a genetic locuswhere editing is desired (e.g., a mutant allele of the GUCY2D gene). Asused herein, “on-target editing,” “on-target activity,” or “on-targetcleavage” means editing at a genetic locus where editing is desired. AR838S gRNA, a R838C gRNA, a R838H gRNA, a double mutation R838CH gRNA,or a double mutation R838SH gRNA has on-target activity when the gRNAdirects editing of the corresponding mutant allele (or mutant alleles inthe case of double mutation gRNAs) at or near the R838 position.

gRNAs of the present disclosure can also direct editing at a geneticlocus where editing is not desired. As used herein, “off-targetediting,” “off-target activity,” or “off-target cleavage” means editingat a genetic locus where editing is not desired.

Off-target editing can be editing of a wild-type allele of the GUCY2Dgene. Herein, this type of off-target editing is termed “wild-typeoff-target editing,” “wild-type off-target activity,” or “wild-typeoff-target cleavage.” A R838S gRNA, a R838C gRNA, a R838H gRNA, a doublemutation R838CH gRNA, or a double mutation R838SH gRNA can havewild-type off-target activity when the gRNA directs editing of awild-type GUCY2D allele.

Off-target editing can be editing of a second gene or locus (e.g.,editing of a genomic sequence that is not a sequence of the GUCY2D geneor a regulatory sequence of the GUCY2D gene). Herein, this type ofoff-target editing is termed “genomic off-target editing,” “genomicoff-target activity,” or “genomic off-target cleavage.” A R838S gRNA, aR838C gRNA, a R838H gRNA, a double mutation R838CH gRNA, or a doublemutation R838SH gRNA has genomic off-target activity when the gRNAdirects editing of a genomic sequence that is not a sequence of theGUCY2D gene or a regulatory sequence of the GUCY2D gene.

In some examples, wild-type off-target activity of a gRNA can be“minimal.” gRNAs with minimal wild-type off-target activity can bedetermined using methods known in the art, for example, methods based onin silico analysis, in vitro methods, or in vivo methods of determiningthe amount of wild-type off-target editing caused by a gRNA. A gRNA withminimal wild-type off-target activity can cause off-target editing in30% or less of cells, for example, 25% or less of cells, 20% or less ofcells, 15% or less of cells 10% or less of cells, 5% or less of cells,4% or less of cells, 3% or less of cells, 2% or less of cells, 1% orless of cells, 0.5% or less of cells, 0.25% or less of cells, or 0.1% orless of cells. Such determinations can, in some cases, be determinedusing in vitro systems.

In some examples, genomic off-target activity of a gRNA can be“minimal.” gRNAs with minimal genomic off-target activity can bedetermined based on in silico analysis, in vitro methods, or in vivomethods of determining the amount of genomic off-target editing causedby a gRNA. A gRNA with minimal genomic off-target activity can cause atleast one instance of genomic off-target editing in 30% or less of cellssuch as, for example, 25% or less of cells, 20% or less of cells, 15% orless of cells 10% or less of cells, 5% or less of cells, 4% or less ofcells, 3% or less of cells, 2% or less of cells, 1% or less of cells,0.5% or less of cells, 0.25% or less of cells, or 0.1% or less of cells.Such determinations can, in some cases, be determined using in vitrosystems.

Another aspect of target sequence selection relates to homologousrecombination events. Sequences sharing regions of homology can serve asfocal points for homologous recombination events that result in deletionof intervening sequences. Such recombination events occur during thenormal course of replication of chromosomes and other DNA sequences, andalso at other times when DNA sequences are being synthesized, such as inthe case of repairs of DSBs, which occur on a regular basis during thenormal cell replication cycle but can also be enhanced by the occurrenceof various events (such as UV light and other inducers of DNA breakage)or the presence of certain agents (such as various chemical inducers).Many such inducers cause DSBs to occur indiscriminately in the genome,and DSBs can be regularly induced and repaired in normal cells. Duringrepair, the original sequence can be reconstructed with completefidelity, however, in some cases, small insertions or deletions(referred to as “indels”) are introduced at the DSB site.

DSBs can also be specifically induced at particular locations, as in thecase of the endonucleases systems described herein, which can be used tocause directed or preferential gene modification events at selectedchromosomal locations. The tendency for homologous sequences to besubject to recombination in the context of DNA repair (as well asreplication) can be taken advantage of in a number of circumstances, andis the basis for one application of gene editing systems, such asCRISPR, in which homology directed repair is used to insert a sequenceof interest, provided through use of a “donor” polynucleotide, into adesired chromosomal location.

Regions of homology between particular sequences, which can be smallregions of “microhomology” that can comprise as few as ten base pairs orless, can also be used to bring about desired deletions. For example, asingle DSB can be introduced at a site that exhibits microhomology witha nearby sequence. During the normal course of repair of such DSB, aresult that occurs with high frequency is the deletion of theintervening sequence as a result of recombination being facilitated bythe DSB and concomitant cellular repair process.

In some circumstances, however, selecting target sequences withinregions of homology can also give rise to much larger deletions,including gene fusions (when the deletions are in coding regions), whichmay or may not be desired given the particular circumstances.

The examples provided herein further illustrate the selection of varioustarget regions for the creation of DSBs designed to induce replacementsthat result in restoration of RetGC1 protein activity, as well as theselection of specific target sequences within such regions that aredesigned to minimize off-target events relative to on-target events.

Homology Direct Repair (HDR)/Donor Nucleotides

Homology direct repair is a cellular mechanism for repairing DSBs. Themost common form is homologous recombination. There are additionalpathways for HDR, including single-strand annealing and alternative-HDR.Genome engineering tools allow researchers to manipulate the cellularhomologous recombination pathways to create site-specific modificationsto the genome. It has been found that cells can repair a double-strandedbreak using a synthetic donor molecule provided in trans. Therefore, byintroducing a double-stranded break near a specific mutation andproviding a suitable donor, targeted changes can be made in the genome.Specific cleavage increases the rate of HDR more than 1,000 fold abovethe rate of 1 in 10⁶ cells receiving a homologous donor alone. The rateof HDR at a particular nucleotide is a function of the distance to thecut site, so choosing overlapping or nearest target sites is important.Gene editing offers the advantage over gene addition, as correcting insitu leaves the rest of the genome unperturbed.

Supplied donors for editing by HDR vary markedly but can contain theintended sequence with small or large flanking homology arms to allowannealing to the genomic DNA. The homology regions flanking theintroduced genetic changes can be 30 bp or smaller, or as large as amulti-kilobase cassette that can contain promoters, cDNAs, etc. Bothsingle-stranded and double-stranded oligonucleotide donors have beenused. These oligonucleotides range in size from less than 100 nt to overmany kb, though longer ssDNA can also be generated and used.Double-stranded donors can be used, including PCR amplicons, plasmids,and mini-circles. In general, it has been found that an AAV vector canbe a very effective means of delivery of a donor template, though thepackaging limits for individual donors is <5 kb. Active transcription ofthe donor increased HDR three-fold, indicating the inclusion of promotermay increase conversion. Conversely, CpG methylation of the donordecreased gene expression and HDR.

Donor nucleotides for correcting mutations often are small (<300 bp).This is advantageous, as HDR efficiencies may be inversely related tothe size of the donor molecule. Also, it is expected that the donortemplates can fit into size constrained AAV molecules, which have beenshown to be an effective means of donor template delivery.

In addition to wildtype endonucleases, such as Cas9, nickase variantsexist that have one or the other nuclease domain inactivated resultingin cutting of only one DNA strand. HDR can be directed from individualCas nickases or using pairs of nickases that flank the target area.Donors can be single-stranded, nicked, or dsDNA.

The donor DNA can be supplied with the nuclease or independently by avariety of different methods, for example by transfection, nanoparticle,microinjection, or viral transduction. A range of tethering options hasbeen proposed to increase the availability of the donors for HDR.Examples include attaching the donor to the nuclease, attaching to DNAbinding proteins that bind nearby, or attaching to proteins that areinvolved in DNA end binding or repair.

The repair pathway choice can be guided by a number of cultureconditions, such as those that influence cell cycling, or by targetingof DNA repair and associated proteins. For example, to increase HDR, keyNHEJ molecules can be suppressed, such as KU70, KU80 or DNA ligase IV.

Without a donor present, the ends from a DNA break or ends fromdifferent breaks can be joined using the several non-homologous repairpathways in which the DNA ends are joined with little or no base-pairingat the junction. In addition to canonical NHEJ, there are similar repairmechanisms, such as ANHEJ. If there are two breaks, the interveningsegment can be deleted or inverted. NHEJ repair pathways can lead toinsertions, deletions or mutations at the joints.

NHEJ was used to insert a 15-kb inducible gene expression cassette intoa defined locus in human cell lines after nuclease cleavage. The methodsof insertion of large inducible gene expression cassettes have beendescribed [Maresca, M., Lin, V. G., Guo, N. & Yang, Y., Genome Res 23,539-546 (2013), Suzuki et al. Nature, 540, 144-149 (2016))].

In addition to genome editing by NHEJ or HDR, site-specific geneinsertions have been conducted that use both the NHEJ pathway and HDR. Acombination approach can be applicable in certain settings, possiblyincluding intron/exon borders. NHEJ may prove effective for ligation inthe intron, while the error-free HDR may be better suited in the codingregion.

Illustrative modifications within the GUCY2D gene include replacementswithin or near (proximal) to the mutations referred to above (i.e.R838H, R838C, or R838S mutations), such as within the region of lessthan 3 kb, less than 2 kb, less than 1 kb, less than 0.5 kb upstream ordownstream of the specific mutation. Given the relatively widevariations of mutations in the GUCY2D gene, it will be appreciated thatnumerous variations of the replacements referenced above (includingwithout limitation larger as well as smaller deletions), would beexpected to result in restoration of the RetGC1 protein activity.

Such variants can include replacements that are larger in the 5′ and/or3′ direction than the specific mutation in question, or smaller ineither direction. Accordingly, by “near” or “proximal” with respect tospecific replacements, it is intended that the SSB or DSB locusassociated with a desired replacement boundary (also referred to hereinas an endpoint) can be within a region that is less than about 3 kb fromthe reference locus, e.g., the mutation site. The SSB or DSB locus canbe more proximal and within 2 kb, within 1 kb, within 0.5 kb, or within0.1 kb. In the case of a small replacement, the desired endpoint can beat or “adjacent to” the reference locus, by which it is intended thatthe endpoint can be within 100 bp, within 50 bp, within 25 bp, or lessthan about 10 bp to 5 bp from the reference locus.

Larger or smaller replacements can provide the same benefit, as long asthe RetGC1 protein activity is restored. It is thus expected that manyvariations of the replacements described and illustrated herein can beeffective for ameliorating autosomal dominant CORD.

The terms “near” or “proximal” with respect to the SSBs or DSBs refer tothe SSBs or DSBs being within 2 kb, within 1 kb, within 0.5 kb, within0.25 kb, within 0.2 kb, or within 0.1 kb of the R838H, R838C, or R838Smutation.

Nucleic Acid Modifications (Chemical and Structural Modifications)

In some cases, polynucleotides introduced into cells can comprise one ormore modifications that can be used individually or in combination, forexample, to enhance activity, stability or specificity, alter delivery,reduce innate immune responses in host cells, or for other enhancements,as further described herein and known in the art.

In certain examples, modified polynucleotides can be used in theCRISPR/Cas9/Cpf1 system, in which case the guide RNAs (eithersingle-molecule guides or double-molecule guides) and/or a DNA or an RNAencoding a Cas or Cpf1 endonuclease introduced into a cell can bemodified, as described and illustrated below. Such modifiedpolynucleotides can be used in the CRISPR/Cas9/Cpf1 system to edit anyone or more genomic loci.

Using the CRISPR/Cas9/Cpf1 system for purposes of non-limitingillustrations of such uses, modifications of guide RNAs can be used toenhance the formation or stability of the CRISPR/Cas9/Cpf1 genomeediting complex comprising guide RNAs, which can be single-moleculeguides or double-molecule, and a Cas or Cpf1 endonuclease. Modificationsof guide RNAs can also or alternatively be used to enhance theinitiation, stability or kinetics of interactions between the genomeediting complex with the target sequence in the genome, which can beused, for example, to enhance on-target activity. Modifications of guideRNAs can also or alternatively be used to enhance specificity, e.g., therelative rates of genome editing at the on-target site as compared toeffects at other (off-target) sites.

Modifications can also or alternatively be used to increase thestability of a guide RNA, e.g., by increasing its resistance todegradation by ribonucleases (RNases) present in a cell, thereby causingits half-life in the cell to be increased. Modifications enhancing guideRNA half-life can be particularly useful in aspects in which a Cas orCpf1 endonuclease is introduced into the cell to be edited via an RNAthat needs to be translated in order to generate endonuclease, becauseincreasing the half-life of guide RNAs introduced at the same time asthe RNA encoding the endonuclease can be used to increase the time thatthe guide RNAs and the encoded Cas or Cpf1 endonuclease co-exist in thecell.

Modifications can also or alternatively be used to decrease thelikelihood or degree to which RNAs introduced into cells elicit innateimmune responses. Such responses, which have been well characterized inthe context of RNA interference (RNAi), including small-interfering RNAs(siRNAs), as described below and in the art, tend to be associated withreduced half-life of the RNA and/or the elicitation of cytokines orother factors associated with immune responses.

One or more types of modifications can also be made to RNAs encoding anendonuclease that are introduced into a cell, including, withoutlimitation, modifications that enhance the stability of the RNA (such asby increasing its degradation by RNAses present in the cell),modifications that enhance translation of the resulting product (i.e.the endonuclease), and/or modifications that decrease the likelihood ordegree to which the RNAs introduced into cells elicit innate immuneresponses.

Combinations of modifications, such as the foregoing and others, canlikewise be used. In the case of CRISPR/Cas9/Cpf1, for example, one ormore types of modifications can be made to guide RNAs (including thoseexemplified above), and/or one or more types of modifications can bemade to RNAs encoding Cas endonuclease (including those exemplifiedabove).

By way of illustration, guide RNAs used in the CRISPR/Cas9/Cpf1 system,or other smaller RNAs can be readily synthesized by chemical means,enabling a number of modifications to be readily incorporated, asillustrated below and described in the art. While chemical syntheticprocedures are continually expanding, purifications of such RNAs byprocedures such as high-performance liquid chromatography (HPLC, whichavoids the use of gels such as PAGE) tends to become more challenging aspolynucleotide lengths increase significantly beyond a hundred or sonucleotides. One approach that can be used for generating chemicallymodified RNAs of greater length is to produce two or more molecules thatare ligated together. Much longer RNAs, such as those encoding a Cas9endonuclease, are more readily generated enzymatically. While fewertypes of modifications are available for use in enzymatically producedRNAs, there are still modifications that can be used to, e.g., enhancestability, reduce the likelihood or degree of innate immune response,and/or enhance other attributes, as described further below and in theart; and new types of modifications are regularly being developed.

By way of illustration of various types of modifications, especiallythose used frequently with smaller chemically synthesized RNAs,modifications can comprise one or more nucleotides modified at the 2′position of the sugar, in some aspects a 2′-O-alkyl, 2′-O-alkyl-O-alkyl,or 2′-fluoro-modified nucleotide. In some examples, RNA modificationscan comprise 2′-fluoro, 2′-amino or 2′-O-methyl modifications on theribose of pyrimidines, abasic residues, or an inverted base at the 3′end of the RNA. Such modifications can be routinely incorporated intooligonucleotides and these oligonucleotides have been shown to have ahigher Tm (i.e., higher target binding affinity) than2′-deoxyoligonucleotides against a given target.

A number of nucleotide and nucleoside modifications have been shown tomake the oligonucleotide into which they are incorporated more resistantto nuclease digestion than the native oligonucleotide; these modifiedoligos survive intact for a longer time than unmodifiedoligonucleotides. Specific examples of modified oligonucleotides includethose comprising modified backbones, for example, phosphorothioates,phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkylintersugar linkages or short chain heteroatomic or heterocyclicintersugar linkages. Some oligonucleotides are oligonucleotides withphosphorothioate backbones and those with heteroatom backbones,particularly CH₂—NH—O—CH₂, CH, ˜N(CH₃)˜O˜CH₂ (known as amethylene(methylimino) or MMI backbone), CH₂—O—N (CH₃)—CH₂,CH₂—N(CH₃)—N(CH₃)—CH₂ and O—N(CH₃)—CH₂—CH₂ backbones, wherein the nativephosphodiester backbone is represented as O—P—O—CH); amide backbones[see De Mesmaeker et al., Ace. Chem. Res., 28:366-374 (1995)];morpholino backbone structures (see Summerton and Weller, U.S. Pat. No.5,034,506); peptide nucleic acid (PNA) backbone (wherein thephosphodiester backbone of the oligonucleotide is replaced with apolyamide backbone, the nucleotides being bound directly or indirectlyto the aza nitrogen atoms of the polyamide backbone, see Nielsen et al.,Science 1991, 254, 1497). Phosphorus-containing linkages include, butare not limited to, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,methyl and other alkyl phosphonates comprising 3′alkylene phosphonatesand chiral phosphonates, phosphinates, phosphoramidates comprising3′-amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thionoalkylphosphonates,thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′linkages, 2′-5′ linked analogs of these, and those having invertedpolarity wherein the adjacent pairs of nucleoside units are linked 3′-5′to 5′-3′ or 2′-5′ to 5′-2′; see U.S. Pat. Nos. 3,687,808; 4,469,863;4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019;5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496;5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306;5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050.

Morpholino-based oligomeric compounds are described in Braasch and DavidCorey, Biochemistry, 41(14): 4503-4510 (2002); Genesis, Volume 30, Issue3, (2001); Heasman, Dev. Biol., 243: 209-214 (2002); Nasevicius et al.,Nat. Genet., 26:216-220 (2000); Lacerra et al., Proc. Natl. Acad. Sci.,97: 9591-9596 (2000); and U.S. Pat. No. 5,034,506, issued Jul. 23, 1991.

Cyclohexenyl nucleic acid oligonucleotide mimetics are described in Wanget al., J. Am. Chem. Soc., 122: 8595-8602 (2000).

Modified oligonucleotide backbones that do not include a phosphorus atomtherein have backbones that are formed by short chain alkyl orcycloalkyl internucleoside linkages, mixed heteroatom and alkyl orcycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These comprisethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S, and CH₂ component parts; see U.S. Pat. Nos. 5,034,506; 5,166,315;5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564;5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307;5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046;5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and5,677,439.

One or more substituted sugar moieties can also be included, e.g., oneof the following at the 2′ position: OH, SH, SCH₃, F, OCN, OCH₃OCH₃,OCH₃O(CH₂)n CH₃, O(CH₂)n NH₂, or O(CH₂)n CH₃, where n is from 1 to about10; C1 to C10 lower alkyl, alkoxyalkoxy, substituted lower alkyl,alkaryl or aralkyl; Cl; Br; CN; CF₃; OCF₃; O—, S—, or N-alkyl; O—, S—,or N-alkenyl; SOCH₃; SO₂CH₃; ONO₂; NO₂; N₃; NH₂; heterocycloalkyl;heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl;an RNA cleaving group; a reporter group; an intercalator; a group forimproving the pharmacokinetic properties of an oligonucleotide; or agroup for improving the pharmacodynamic properties of an oligonucleotideand other substituents having similar properties. In some aspects, amodification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as2′-O-(2-methoxyethyl)) (Martin et al, HeIv. Chim. Acta, 1995, 78, 486).Other modifications include 2′-methoxy (2′-O—CH₃), 2′-propoxy(2′-OCH₂CH₂CH₃) and 2′-fluoro (2′-F). Similar modifications can also bemade at other positions on the oligonucleotide, particularly the 3′position of the sugar on the 3′ terminal nucleotide and the 5′ positionof 5′ terminal nucleotide. Oligonucleotides can also have sugarmimetics, such as cyclobutyls in place of the pentofuranosyl group.

In some examples, both a sugar and an internucleoside linkage, i.e., thebackbone, of the nucleotide units can be replaced with novel groups. Thebase units can be maintained for hybridization with an appropriatenucleic acid target compound. One such oligomeric compound, anoligonucleotide mimetic that has been shown to have excellenthybridization properties, is referred to as a peptide nucleic acid(PNA). In PNA compounds, the sugar-backbone of an oligonucleotide can bereplaced with an amide containing backbone, for example, anaminoethylglycine backbone. The nucleobases can be retained and bounddirectly or indirectly to aza nitrogen atoms of the amide portion of thebackbone. Representative U.S. patents that teach the preparation of PNAcompounds comprise, but are not limited to, U.S. Pat. Nos. 5,539,082;5,714,331; and 5,719,262. Further teaching of PNA compounds can be foundin Nielsen et al, Science, 254: 1497-1500 (1991).

Guide RNAs can also include, additionally or alternatively, nucleobase(often referred to in the art simply as “base”) modifications orsubstitutions. As used herein, “unmodified” or “natural” nucleobasesinclude adenine (A), guanine (G), thymine (T), cytosine (C), and uracil(U). Modified nucleobases include nucleobases found only infrequently ortransiently in natural nucleic acids, e.g., hypoxanthine,6-methyladenine, 5-Me pyrimidines, particularly 5-methylcytosine (alsoreferred to as 5-methyl-2′ deoxycytosine and often referred to in theart as 5-Me-C), 5-hydroxymethylcytosine (HMC), glycosyl HMC andgentobiosyl HMC, as well as synthetic nucleobases, e.g., 2-aminoadenine,2-(methylamino) adenine, 2-(imidazolylalkyl)adenine, 2-(aminoalklyamino)adenine or other heterosubstituted alkyladenines, 2-thiouracil,2-thiothymine, 5-bromouracil, 5-hydroxymethyluracil, 8-azaguanine,7-deazaguanine, N6 (6-aminohexyl) adenine, and 2,6-diaminopurine.Kornberg, A., DNA Replication, W. H. Freeman & Co., San Francisco, pp.75-77 (1980); Gebeyehu et al., Nucl. Acids Res. 15:4513 (1997). A“universal” base known in the art, e.g., inosine, can also be included.5-Me-C substitutions have been shown to increase nucleic acid duplexstability by 0.6-1.2° C. (Sanghvi, Y. S., in Crooke, S. T. and Lebleu,B., eds., Antisense Research and Applications, CRC Press, Boca Raton,1993, pp. 276-278) and are aspects of base substitutions.

Modified nucleobases can comprise other synthetic and naturalnucleobases, such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and otheralkyl derivatives of adenine and guanine, 2-propyl and other alkylderivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil andcytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudo-uracil),4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl andother 8-substituted adenines and guanines, 5-halo particularly 5-bromo,5-trifluoromethyl and other 5-substituted uracils and cytosines,7-methylquanine and 7-methyladenine, 8-azaguanine and 8-azaadenine,7-deazaguanine and 7-deazaadenine, and 3-deazaguanine and3-deazaadenine.

Further, nucleobases can comprise those disclosed in U.S. Pat. No.3,687,808, those disclosed in ‘The Concise Encyclopedia of PolymerScience and Engineering’, pages 858-859, Kroschwitz, J. I., ed. JohnWiley & Sons, 1990, those disclosed by Englisch et al., AngewandleChemie, International Edition’, 1991, 30, page 613, and those disclosedby Sanghvi, Y. S., Chapter 15, Antisense Research and Applications’,pages 289-302, Crooke, S. T. and Lebleu, B. ea., CRC Press, 1993.Certain of these nucleobases are particularly useful for increasing thebinding affinity of the oligomeric compounds of the invention. Theseinclude 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6substituted purines, comprising 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y.S., Crooke, S. T. and Lebleu, B., eds, ‘Antisense Research andApplications’, CRC Press, Boca Raton, 1993, pp. 276-278) and are aspectsof base substitutions, even more particularly when combined with2′-O-methoxyethyl sugar modifications. Modified nucleobases aredescribed in U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos.4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272;5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540;5,587,469; 5,596,091; 5,614,617; 5,681,941; 5,750,692; 5,763,588;5,830,653; 6,005,096; and U.S. Patent Application Publication2003/0158403.

Thus, the term “modified” refers to a non-natural sugar, phosphate, orbase that is incorporated into a guide RNA, an endonuclease, or both aguide RNA and an endonuclease. It is not necessary for all positions ina given oligonucleotide to be uniformly modified, and in fact more thanone of the aforementioned modifications can be incorporated in a singleoligonucleotide, or even in a single nucleoside within anoligonucleotide.

The guide RNAs and/or mRNA (or DNA) encoding an endonuclease can bechemically linked to one or more moieties or conjugates that enhance theactivity, cellular distribution, or cellular uptake of theoligonucleotide. Such moieties comprise, but are not limited to, lipidmoieties such as a cholesterol moiety [Letsinger et al., Proc. Natl.Acad. Sci. USA, 86: 6553-6556 (1989)]; cholic acid [Manoharan et al.,Bioorg. Med. Chem. Let., 4: 1053-1060 (1994)]; a thioether, e.g.,hexyl-S-tritylthiol [Manoharan et al, Ann. N. Y. Acad. Sci., 660:306-309 (1992) and Manoharan et al., Bioorg. Med. Chem. Let., 3:2765-2770 (1993)]; a thiocholesterol [Oberhauser et al., Nucl. AcidsRes., 20: 533-538 (1992)]; an aliphatic chain, e.g., dodecandiol orundecyl residues [Kabanov et al., FEBS Lett., 259: 327-330 (1990) andSvinarchuk et al., Biochimie, 75: 49-54 (1993)]; a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate [Manoharan et al.,Tetrahedron Lett., 36: 3651-3654 (1995) and Shea et al., Nucl. AcidsRes., 18: 3777-3783 (1990)]; a polyamine or a polyethylene glycol chain[Mancharan et al., Nucleosides & Nucleotides, 14: 969-973 (1995)];adamantane acetic acid [Manoharan et al., Tetrahedron Lett., 36:3651-3654 (1995)]; a palmityl moiety [(Mishra et al., Biochim. Biophys.Acta, 1264: 229-237 (1995)]; or an octadecylamine orhexylamino-carbonyl-t oxycholesterol moiety [Crooke et al., J.Pharmacol. Exp. Ther., 277: 923-937 (1996)]. See also U.S. Pat. Nos.4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717; 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241; 5,391,723; 5,416,203; 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599, 928 and 5,688,941.

Sugars and other moieties can be used to target proteins and complexescomprising nucleotides, such as cationic polysomes and liposomes, toparticular sites. For example, hepatic cell directed transfer can bemediated via asialoglycoprotein receptors (ASGPRs); see, e.g., Hu, etal., Protein Pept Lett. 21(10):1025-30 (2014). Other systems known inthe art and regularly developed can be used to target biomolecules ofuse in the present case and/or complexes thereof to particular targetcells of interest.

These targeting moieties or conjugates can include conjugate groupscovalently bound to functional groups, such as primary or secondaryhydroxyl groups. Conjugate groups of the present disclosure includeintercalators, reporter molecules, polyamines, polyamides, polyethyleneglycols, polyethers, groups that enhance the pharmacodynamic propertiesof oligomers, and groups that enhance the pharmacokinetic properties ofoligomers. Typical conjugate groups include cholesterols, lipids,phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone,acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups thatenhance the pharmacodynamic properties, in the context of this presentdisclosure, include groups that improve uptake, enhance resistance todegradation, and/or strengthen sequence-specific hybridization with thetarget nucleic acid. Groups that enhance the pharmacokinetic properties,in the context of this invention, include groups that improve uptake,distribution, metabolism or excretion of the compounds of the presentdisclosure. Representative conjugate groups are disclosed inInternational Patent Application No. PCT/US92/09196, filed Oct. 23, 1992(published as WO1993007883), and U.S. Pat. No. 6,287,860. Conjugatemoieties include, but are not limited to, lipid moieties such as acholesterol moiety, cholic acid, a thioether, e.g., hexyl-5-tritylthiol,a thiocholesterol, an aliphatic chain, e.g., dodecandiol or undecylresidues, a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, apolyamine or a polyethylene glycol chain, or adamantane acetic acid, apalmityl moiety, or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety. See, e.g., U.S. Pat. Nos. 4,828,979; 4,948,882;5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717,5,580,731; 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045;5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044;4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263;4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136;5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506;5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241; 5,391,723;5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552;5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696;5,599,923; 5,599,928 and 5,688,941.

Longer polynucleotides that are less amenable to chemical synthesis andare typically produced by enzymatic synthesis can also be modified byvarious means. Such modifications can include, for example, theintroduction of certain nucleotide analogs, the incorporation ofparticular sequences or other moieties at the 5′ or 3′ ends ofmolecules, and other modifications. By way of illustration, the mRNAencoding Cas9 is approximately 4 kb in length and can be synthesized byin vitro transcription. Modifications to the mRNA can be applied to,e.g., increase its translation or stability (such as by increasing itsresistance to degradation with a cell), or to reduce the tendency of theRNA to elicit an innate immune response that is often observed in cellsfollowing introduction of exogenous RNAs, particularly longer RNAs suchas that encoding Cas9.

Numerous such modifications have been described in the art, such aspolyA tails, 5′ cap analogs (e.g., Anti Reverse Cap Analog (ARCA) orm7G(5′)ppp(5′)G (mCAP)), modified 5′ or 3′ untranslated regions (UTRs),use of modified bases (such as Pseudo-UTP, 2-Thio-UTP,5-Methylcytidine-5′-Triphosphate (5-Methyl-CTP) or N6-Methyl-ATP), ortreatment with phosphatase to remove 5′ terminal phosphates. These andother modifications are known in the art, and new modifications of RNAsare regularly being developed.

There are numerous commercial suppliers of modified RNAs, including forexample, TriLink Biotech, AxoLabs, Bio-Synthesis Inc., Dharmacon andmany others. As described by TriLink, for example, 5-Methyl-CTP can beused to impart desirable characteristics, such as increased nucleasestability, increased translation or reduced interaction of innate immunereceptors with in vitro transcribed RNA.5-Methylcytidine-5′-Triphosphate (5-Methyl-CTP), N6-Methyl-ATP, as wellas Pseudo-UTP and 2-Thio-UTP, have also been shown to reduce innateimmune stimulation in culture and in vivo while enhancing translation,as illustrated in publications by Kormann et al. and Warren et al.referred to below.

It has been shown that chemically modified mRNA delivered in vivo can beused to achieve improved therapeutic effects; see, e.g., Kormann et al.,Nature Biotechnology 29, 154-157 (2011). Such modifications can be used,for example, to increase the stability of the RNA molecule and/or reduceits immunogenicity. Using chemical modifications such as Pseudo-U,N6-Methyl-A, 2-Thio-U and 5-Methyl-C, it was found that substitutingjust one quarter of the uridine and cytidine residues with 2-Thio-U and5-Methyl-C respectively resulted in a significant decrease in toll-likereceptor (TLR) mediated recognition of the mRNA in mice. By reducing theactivation of the innate immune system, these modifications can be usedto effectively increase the stability and longevity of the mRNA in vivo;see, e.g., Kormann et al., supra.

It has also been shown that repeated administration of syntheticmessenger RNAs incorporating modifications designed to bypass innateanti-viral responses can reprogram differentiated human cells topluripotency. See, e.g., Warren, et al., Cell Stem Cell, 7(5):618-30(2010). Such modified mRNAs that act as primary reprogramming proteinscan be an efficient means of reprogramming multiple human cell types.Such cells are referred to as induced pluripotency stem cells (iPSCs),and it was found that enzymatically synthesized RNA incorporating5-Methyl-CTP, Pseudo-UTP and an Anti Reverse Cap Analog (ARCA) could beused to effectively evade the cell's antiviral response; see, e.g.,Warren et al., supra.

Other modifications of polynucleotides described in the art include, forexample, the use of polyA tails, the addition of 5′ cap analogs such as(m7G(5′)ppp(5′)G (mCAP)), modifications of 5′ or 3′ untranslated regions(UTRs), or treatment with phosphatase to remove 5′ terminalphosphates—and new approaches are regularly being developed.

A number of compositions and techniques applicable to the generation ofmodified RNAs for use herein have been developed in connection with themodification of RNA interference (RNAi), including small-interferingRNAs (siRNAs). siRNAs present particular challenges in vivo becausetheir effects on gene silencing via mRNA interference are generallytransient, which can require repeat administration. In addition, siRNAsare double-stranded RNAs (dsRNA) and mammalian cells have immuneresponses that have evolved to detect and neutralize dsRNA, which isoften a by-product of viral infection. Thus, there are mammalian enzymessuch as PKR (dsRNA-responsive kinase), and potentially retinoicacid-inducible gene I (RIG-I), that can mediate cellular responses todsRNA, as well as Toll-like receptors (such as TLR3, TLR7 and TLR8) thatcan trigger the induction of cytokines in response to such molecules;see, e.g., the reviews by Angart et al., Pharmaceuticals (Basel) 6(4):440-468 (2013); Kanasty et al., Molecular Therapy 20(3): 513-524 (2012);Burnett et al., Biotechnol J. 6(9):1130-46 (2011); Judge and MacLachlan,Hum Gene Ther 19(2):111-24 (2008).

A large variety of modifications have been developed and applied toenhance RNA stability, reduce innate immune responses, and/or achieveother benefits that can be useful in connection with the introduction ofpolynucleotides into human cells, as described herein; see, e.g., thereviews by Whitehead K A et al., Annual Review of Chemical andBiomolecular Engineering, 2: 77-96 (2011); Gaglione and Messere, MiniRev Med Chem, 10(7):578-95 (2010); Chernolovskaya et al, Curr Opin MolTher., 12(2):158-67 (2010); Deleavey et al., Curr Protoc Nucleic AcidChem Chapter 16:Unit 16.3 (2009); Behlke, Oligonucleotides 18(4):305-19(2008); Fucini et al., Nucleic Acid Ther 22(3): 205-210 (2012); Bremsenet al., Front Genet 3:154 (2012).

As noted above, there are a number of commercial suppliers of modifiedRNAs, many of which have specialized in modifications designed toimprove the effectiveness of siRNAs. A variety of approaches are offeredbased on various findings reported in the literature. For example,Dharmacon notes that replacement of a non-bridging oxygen with sulfur(phosphorothioate, PS) has been extensively used to improve nucleaseresistance of siRNAs, as reported by Kole, Nature Reviews Drug Discovery11:125-140 (2012). Modifications of the 2′-position of the ribose havebeen reported to improve nuclease resistance of the internucleotidephosphate bond while increasing duplex stability (Tm), which has alsobeen shown to provide protection from immune activation. A combinationof moderate PS backbone modifications with small, well-tolerated2′-substitutions (2′-O-Methyl, 2′-Fluoro, 2′-Hydro) have been associatedwith highly stable siRNAs for applications in vivo, as reported bySoutschek et al. Nature 432:173-178 (2004); and 2′-O-Methylmodifications have been reported to be effective in improving stabilityas reported by Volkov, Oligonucleotides 19:191-202 (2009). With respectto decreasing the induction of innate immune responses, modifyingspecific sequences with 2′-O-Methyl, 2′-Fluoro, 2′-Hydro have beenreported to reduce TLR7/TLR8 interaction while generally preservingsilencing activity; see, e.g., Judge et al., Mol. Ther. 13:494-505(2006); and Cekaite et al., J. Mol. Biol. 365:90-108 (2007). Additionalmodifications, such as 2-thiouracil, pseudouracil, 5-methylcytosine,5-methyluracil, and N6-methyladenosine have also been shown to minimizethe immune effects mediated by TLR3, TLR7, and TLR8; see, e.g., Kariko,K. et al., Immunity 23:165-175 (2005).

As is also known in the art, and commercially available, a number ofconjugates can be applied to polynucleotides, such as RNAs, for useherein that can enhance their delivery and/or uptake by cells, includingfor example, cholesterol, tocopherol and folic acid, lipids, peptides,polymers, linkers and aptamers; see, e.g., the review by Winkler, Ther.Deliv. 4:791-809 (2013).

Codon-Optimization

A polynucleotide encoding a site-directed polypeptide can becodon-optimized according to methods standard in the art for expressionin the cell containing the target DNA of interest. For example, if theintended target nucleic acid is in a human cell, a human codon-optimizedpolynucleotide encoding Cas9 is contemplated for use for producing theCas9 polypeptide.

Nucleic Acids Encoding System Components

The present disclosure provides a nucleic acid comprising a nucleotidesequence encoding a genome-targeting nucleic acid of the disclosure, asite-directed polypeptide of the disclosure, and/or any nucleic acid orproteinaceous molecule necessary to carry out the aspects of the methodsof the disclosure.

The nucleic acid encoding a genome-targeting nucleic acid of thedisclosure, a site-directed polypeptide of the disclosure, and/or anynucleic acid or proteinaceous molecule necessary to carry out theaspects of the methods of the disclosure can comprise a vector (e.g., arecombinant expression vector).

The term “vector” refers to a nucleic acid molecule capable oftransporting another nucleic acid to which it has been linked. One typeof vector is a “plasmid”, which refers to a circular double-stranded DNAloop into which additional nucleic acid segments can be ligated.

Another type of vector is a viral vector; wherein additional nucleicacid segments can be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome.

In some examples, vectors can be capable of directing the expression ofnucleic acids to which they are operatively linked. Such vectors arereferred to herein as “recombinant expression vectors”, or more simply“expression vectors”, which serve equivalent functions.

The term “operably linked” means that the nucleotide sequence ofinterest is linked to regulatory sequence(s) in a manner that allows forexpression of the nucleotide sequence. The term “regulatory sequence” isintended to include, for example, promoters, enhancers and otherexpression control elements (e.g., polyadenylation signals). Suchregulatory sequences are well known in the art and are described, forexample, in Goeddel; Gene Expression Technology: Methods in Enzymology185, Academic Press, San Diego, Calif. (1990). Regulatory sequencesinclude those that direct constitutive expression of a nucleotidesequence in many types of host cells, and those that direct expressionof the nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). It will be appreciated by thoseskilled in the art that the design of the expression vector can dependon such factors as the choice of the target cell, the level ofexpression desired, and the like.

Expression vectors contemplated include, but are not limited to, viralvectors based on vaccinia virus, poliovirus, adenovirus,adeno-associated virus, SV40, herpes simplex virus, humanimmunodeficiency virus, retrovirus (e.g., Murine Leukemia Virus, spleennecrosis virus, and vectors derived from retroviruses such as RousSarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus,human immunodeficiency virus, myeloproliferative sarcoma virus, andmammary tumor virus) and other recombinant vectors. Other vectorscontemplated for eukaryotic target cells include, but are not limitedto, the vectors pXT1, pSG5, pSVK3, pBPV, pMSG, and pSVLSV40 (Pharmacia).Additional vectors contemplated for eukaryotic target cells include, butare not limited to, the vectors pCTx-1, pCTx-2, and pCTx-3. Othervectors can be used so long as they are compatible with the host cell.

In some examples, a vector can comprise one or more transcription and/ortranslation control elements. Depending on the host/vector systemutilized, any of a number of suitable transcription and translationcontrol elements, including constitutive and inducible promoters,transcription enhancer elements, transcription terminators, etc. can beused in the expression vector. The vector can be a self-inactivatingvector that either inactivates the viral sequences or the components ofthe CRISPR machinery or other elements.

Non-limiting examples of suitable eukaryotic promoters (i.e., promotersfunctional in a eukaryotic cell) include those from cytomegalovirus(CMV) immediate early, herpes simplex virus (HSV) thymidine kinase,early and late SV40, long terminal repeats (LTRs) from retrovirus, humanelongation factor-1 promoter (EF1), a hybrid construct comprising thecytomegalovirus (CMV) enhancer fused to the chicken beta-actin promoter(CAG), murine stem cell virus promoter (MSCV), phosphoglycerate kinase-1locus promoter (PGK), and mouse metallothionein-I.

For expressing small RNAs, including guide RNAs used in connection withCas endonuclease, various promoters such as RNA polymerase IIIpromoters, including for example U6 and H1, can be advantageous.Descriptions of and parameters for enhancing the use of such promotersare known in art, and additional information and approaches areregularly being described; see, e.g., Ma, H. et al., MolecularTherapy—Nucleic Acids 3, e161 (2014) doi:10.1038/mtna.2014.12.

The expression vector can also contain a ribosome binding site fortranslation initiation and a transcription terminator. The expressionvector can also comprise appropriate sequences for amplifyingexpression. The expression vector can also include nucleotide sequencesencoding non-native tags (e.g., histidine tag, hemagglutinin tag, greenfluorescent protein, etc.) that are fused to the site-directedpolypeptide, thus resulting in a fusion protein.

A promoter can be an inducible promoter (e.g., a heat shock promoter,tetracycline-regulated promoter, steroid-regulated promoter,metal-regulated promoter, estrogen receptor-regulated promoter, etc.).The promoter can be a constitutive promoter (e.g., CMV promoter, UBCpromoter). In some cases, the promoter can be a spatially restrictedand/or temporally restricted promoter (e.g., a tissue specific promoter,a cell type specific promoter, etc.).

The nucleic acid encoding a genome-targeting nucleic acid of thedisclosure and/or a site-directed polypeptide can be packaged into or onthe surface of delivery vehicles for delivery to cells. Deliveryvehicles contemplated include, but are not limited to, nanospheres,liposomes, quantum dots, nanoparticles, polyethylene glycol particles,hydrogels, and micelles. As described in the art, a variety of targetingmoieties can be used to enhance the preferential interaction of suchvehicles with desired cell types or locations.

Introduction of the complexes, polypeptides, and nucleic acids of thedisclosure into cells can occur by viral or bacteriophage infection,transfection, conjugation, protoplast fusion, lipofection,electroporation, nucleofection, calcium phosphate precipitation,polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediatedtransfection, liposome-mediated transfection, particle gun technology,calcium phosphate precipitation, direct micro-injection,nanoparticle-mediated nucleic acid delivery, and the like.

MicroRNAs (MiRNAs)

Another class of gene regulatory regions is microRNA (miRNA) bindingsites. miRNAs are non-coding RNAs that play key roles inpost-transcriptional gene regulation. miRNAs can regulate the expressionof 30% of all mammalian protein-encoding genes. Specific and potent genesilencing by double stranded RNA (RNAi) was discovered, plus additionalsmall noncoding RNA (Canver, M. C. et al., Nature (2015)). The largestclass of non-coding RNAs important for gene silencing is miRNAs. Inmammals, miRNAs are first transcribed as long RNA transcripts, which canbe separate transcriptional units, part of protein introns, or othertranscripts. The long transcripts are called primary miRNA (pri-miRNA)that include imperfectly base-paired hairpin structures. These pri-miRNAcan be cleaved into one or more shorter precursor miRNAs (pre-miRNAs) byMicroprocessor, a protein complex in the nucleus, involving Drosha.

Pre-miRNAs are short stem loops ˜70 nucleotides in length with a2-nucleotide 3′-overhang that are exported into the mature 19-25nucleotide miRNA:miRNA* duplexes. The miRNA strand with lower basepairing stability (the guide strand) can be loaded onto the RNA-inducedsilencing complex (RISC). The passenger guide strand (marked with *),can be functional, but is usually degraded. The mature miRNA tethersRISC to partly complementary sequence motifs in target mRNAspredominantly found within the 3′ untranslated regions (UTRs) andinduces posttranscriptional gene silencing (Bartel, D. P. Cell 136,215-233 (2009); Saj, A. & Lai, E. C. Curr Opin Genet Dev 21, 504-510(2011)).

miRNAs can be important in development, differentiation, cell cycle andgrowth control, and in virtually all biological pathways in mammals andother multicellular organisms. miRNAs can also be involved in cell cyclecontrol, apoptosis and stem cell differentiation, hematopoiesis,hypoxia, muscle development, neurogenesis, insulin secretion,cholesterol metabolism, aging, viral replication and immune responses.

A single miRNA can target hundreds of different mRNA transcripts, whilean individual transcript can be targeted by many different miRNAs. Morethan 28645 miRNAs have been annotated in the latest release of miRBase(v.21). Some miRNAs can be encoded by multiple loci, some of which canbe expressed from tandemly co-transcribed clusters. The features allowfor complex regulatory networks with multiple pathways and feedbackcontrols. miRNAs can be integral parts of these feedback and regulatorycircuits and can help regulate gene expression by keeping proteinproduction within limits (Herranz, H. & Cohen, S. M. Genes Dev 24,1339-1344 (2010); Posadas, D. M. & Carthew, R. W. Curr Opin Genet Dev27, 1-6 (2014)).

miRNAs can also be important in a large number of human diseases thatare associated with abnormal miRNA expression. This associationunderscores the importance of the miRNA regulatory pathway. Recent miRNAdeletion studies have linked miRNAs with regulation of the immuneresponses (Stern-Ginossar, N. et al., Science 317, 376-381 (2007)).

miRNAs also have a strong link to cancer and can play a role indifferent types of cancer. miRNAs have been found to be downregulated ina number of tumors. miRNAs can be important in the regulation of keycancer-related pathways, such as cell cycle control and the DNA damageresponse, and can therefore be used in diagnosis and can be targetedclinically. miRNAs can delicately regulate the balance of angiogenesis,such that experiments depleting all miRNAs suppress tumor angiogenesis(Chen, S. et al., Genes Dev 28, 1054-1067 (2014)).

As has been shown for protein coding genes, miRNA genes can also besubject to epigenetic changes occurring with cancer. Many miRNA loci canbe associated with CpG islands increasing their opportunity forregulation by DNA methylation (Weber, B., Stresemann, C., Brueckner, B.& Lyko, F. Cell Cycle 6, 1001-1005 (2007)). The majority of studies haveused treatment with chromatin remodeling drugs to reveal epigeneticallysilenced miRNAs.

In addition to their role in RNA silencing, miRNAs can also activatetranslation (Posadas, D. M. & Carthew, R. W. Curr Opin Genet Dev 27, 1-6(2014)). Knocking out these sites can lead to decreased expression ofthe targeted gene, while introducing these sites can increaseexpression.

Individual miRNAs can be knocked out most effectively by mutating theseed sequence (bases 2-8 of the miRNA), which can be important forbinding specificity. Cleavage in this region, followed by mis-repair byNHEJ can effectively abolish miRNA function by blocking binding totarget sites. miRNAs could also be inhibited by specific targeting ofthe special loop region adjacent to the palindromic sequence.Catalytically inactive Cas9 can also be used to inhibit shRNA expression(Zhao, Y. et al., Sci Rep 4, 3943 (2014)). In addition to targeting themiRNAs, the binding sites can also be targeted and mutated to preventthe silencing by miRNAs.

According to the present disclosure, any of the miRNAs or their bindingsites can be incorporated into the compositions of the invention.

The compositions can have a region such as, but not limited to, a regioncomprising the sequence of any of the miRNAs listed in SEQ ID NOs:613-4696, the reverse complement of the miRNAs listed in SEQ ID NOs:613-4696, or the miRNA anti-seed region of any of the miRNAs listed inSEQ ID NOs: 613-4696.

The compositions of the invention can comprise one or more miRNA targetsequences, miRNA sequences, or miRNA seeds. Such sequences cancorrespond to any known miRNA such as those taught in US Publication No.2005/0261218 and US Publication No. 2005/0059005. As a non-limitingexample, known miRNAs, their sequences, and their binding site sequencesin the human genome are listed in SEQ ID NOs: 613-4696.

A miRNA sequence comprises a “seed” region, i.e., a sequence in theregion of positions 2-8 of the mature miRNA, which sequence has perfectWatson-Crick complementarity to the miRNA target sequence. A miRNA seedcan comprise positions 2-8 or 2-7 of the mature miRNA. In some examples,a miRNA seed can comprise 7 nucleotides (e.g., nucleotides 2-8 of themature miRNA), wherein the seed-complementary site in the correspondingmiRNA target is flanked by an adenine (A) opposed to miRNA position 1.In some examples, a miRNA seed can comprise 6 nucleotides (e.g.,nucleotides 2-7 of the mature miRNA), wherein the seed-complementarysite in the corresponding miRNA target is flanked by an adenine (A)opposed to miRNA position 1. See for example, Grimson A, Farh K K,Johnston W K, Garrett-Engele P, Lim L P, Bartel D P; Mol Cell. 2007 Jul.6; 27(1):91-105. The bases of the miRNA seed have completecomplementarity with the target sequence.

Identification of miRNAs, miRNA target regions, and their expressionpatterns and role in biology have been reported (Bonauer et al., CurrDrug Targets 2010 11:943-949; Anand and Cheresh Curr Opin Hematol 201118:171-176; Contreras and Rao Leukemia 2012 26:404-413 (2011 Dec. 20.doi: 10.1038/1eu.2011.356); Bartel Cell 2009 136:215-233; Landgraf etal, Cell, 2007 129:1401-1414; Gentner and Naldini, Tissue Antigens. 201280:393-403.

For example, if the composition is not intended to be delivered to theliver but ends up there, then miR-122, a miRNA abundant in liver, caninhibit the expression of the sequence delivered if one or multipletarget sites of miR-122 are engineered into the polynucleotide encodingthat target sequence. Introduction of one or multiple binding sites fordifferent miRNAs can be engineered to further decrease the longevity,stability, and protein translation hence providing an additional layerof tenability.

As used herein, the term “miRNA site” refers to a miRNA target site or amiRNA recognition site, or any nucleotide sequence to which a miRNAbinds or associates. It should be understood that “binding” can followtraditional Watson-Crick hybridization rules or can reflect any stableassociation of the miRNA with the target sequence at or adjacent to themiRNA site.

Conversely, for the purposes of the compositions of the presentdisclosure, miRNA binding sites can be engineered out of (i.e. removedfrom) sequences in which they naturally occur in order to increaseprotein expression in specific tissues. For example, miR-122 bindingsites can be removed to improve protein expression in the liver.

Specifically, miRNAs are known to be differentially expressed in immunecells (also called hematopoietic cells), such as antigen presentingcells (APCs) (e.g. dendritic cells and macrophages), macrophages,monocytes, B lymphocytes, T lymphocytes, granulocytes, natural killercells, etc. Immune cell specific miRNAs are involved in immunogenicity,autoimmunity, the immune-response to infection, inflammation, as well asunwanted immune response after gene therapy and tissue/organtransplantation. Immune cells specific miRNAs also regulate many aspectsof development, proliferation, differentiation and apoptosis ofhematopoietic cells (immune cells). For example, miR-142 and miR-146 areexclusively expressed in the immune cells, particularly abundant inmyeloid dendritic cells. Introducing the miR-142 binding site into the3′-UTR of a polypeptide of the present disclosure can selectivelysuppress the gene expression in the antigen presenting cells throughmiR-142 mediated mRNA degradation, limiting antigen presentation inprofessional APCs (e.g. dendritic cells) and thereby preventingantigen-mediated immune response after gene delivery (see, Annoni A etal., blood, 2009, 114, 5152-5161.

In one example, miRNA binding sites that are known to be expressed inimmune cells, in particular, the antigen presenting cells, can beengineered into the polynucleotides to suppress the expression of thepolynucleotide in APCs through miRNA mediated RNA degradation, subduingthe antigen-mediated immune response, while the expression of thepolynucleotide is maintained in non-immune cells where the immune cellspecific miRNAs are not expressed.

Many miRNA expression studies have been conducted, and are described inthe art, to profile the differential expression of miRNAs in variouscancer cells/tissues and other diseases. Some miRNAs are abnormallyover-expressed in certain cancer cells and others are under-expressed.For example, miRNAs are differentially expressed in cancer cells(WO2008/154098, US2013/0059015, US2013/0042333, WO2011/157294); cancerstem cells (US2012/0053224); pancreatic cancers and diseases(US2009/0131348, US2011/0171646, US2010/0286232, U.S. Pat. No.8,389,210); asthma and inflammation (U.S. Pat. No. 8,415,096); prostatecancer (US2013/0053264); hepatocellular carcinoma (WO2012/151212,US2012/0329672, WO2008/054828, U.S. Pat. No. 8,252,538); lung cancercells (WO2011/076143, WO2013/033640, WO2009/070653, US2010/0323357);cutaneous T-cell lymphoma (WO2013/011378); colorectal cancer cells(WO2011/0281756, WO2011/076142); cancer positive lymph nodes(WO2009/100430, US2009/0263803); nasopharyngeal carcinoma (EP2112235);chronic obstructive pulmonary disease (US2012/0264626, US2013/0053263);thyroid cancer (WO2013/066678); ovarian cancer cells (US2012/0309645,WO2011/095623); breast cancer cells (WO2008/154098, WO2007/081740,US2012/0214699), leukemia and lymphoma (WO2008/073915, US2009/0092974,US2012/0316081, US2012/0283310, WO2010/018563.

Human Cells

For ameliorating autosomal dominant CORD or any disorder associated withGUCY2D, as described and illustrated herein, the principal targets forgene editing are human cells. For example, in the ex vivo methods, thehuman cells can be somatic cells, which after being modified using thetechniques as described, can give rise to differentiated cells, e.g.,photoreceptor cells or retinal progenitor cells. For example, in the invivo methods, the human cells can be photoreceptor cells or retinalprogenitor cells.

By performing gene editing in autologous cells that are derived from andtherefore already completely matched with the patient in need, it ispossible to generate cells that can be safely re-introduced into thepatient, and effectively give rise to a population of cells that can beeffective in ameliorating one or more clinical conditions associatedwith the patient's disease.

Progenitor cells (also referred to as stem cells herein) are capable ofboth proliferation and giving rise to more progenitor cells, these inturn having the ability to generate a large number of mother cells thatcan in turn give rise to differentiated or differentiable daughtercells. The daughter cells themselves can be induced to proliferate andproduce progeny that subsequently differentiate into one or more maturecell types, while also retaining one or more cells with parentaldevelopmental potential. The term “stem cell” refers then, to a cellwith the capacity or potential, under particular circumstances, todifferentiate to a more specialized or differentiated phenotype, andwhich retains the capacity, under certain circumstances, to proliferatewithout substantially differentiating. In one aspect, the termprogenitor or stem cell refers to a generalized mother cell whosedescendants (progeny) specialize, often in different directions, bydifferentiation, e.g., by acquiring completely individual characters, asoccurs in progressive diversification of embryonic cells and tissues.Cellular differentiation is a complex process typically occurringthrough many cell divisions. A differentiated cell can derive from amultipotent cell that itself is derived from a multipotent cell, and soon. While each of these multipotent cells can be considered stem cells,the range of cell types that each can give rise to can varyconsiderably. Some differentiated cells also have the capacity to giverise to cells of greater developmental potential. Such capacity can benatural or can be induced artificially upon treatment with variousfactors. In many biological instances, stem cells can also be“multipotent” because they can produce progeny of more than one distinctcell type, but this is not required for “stem-ness.”

Self-renewal can be another important aspect of the stem cell. Intheory, self-renewal can occur by either of two major mechanisms. Stemcells can divide asymmetrically, with one daughter retaining the stemstate and the other daughter expressing some distinct other specificfunction and phenotype. Alternatively, some of the stem cells in apopulation can divide symmetrically into two stems, thus maintainingsome stem cells in the population as a whole, while other cells in thepopulation give rise to differentiated progeny only. Generally,“progenitor cells” have a cellular phenotype that is more primitive(i.e., is at an earlier step along a developmental pathway orprogression than is a fully differentiated cell). Often, progenitorcells also have significant or very high proliferative potential.Progenitor cells can give rise to multiple distinct differentiated celltypes or to a single differentiated cell type, depending on thedevelopmental pathway and on the environment in which the cells developand differentiate.

In the context of cell ontogeny, the adjective “differentiated,” or“differentiating” is a relative term. A “differentiated cell” is a cellthat has progressed further down the developmental pathway than the cellto which it is being compared. Thus, stem cells can differentiate intolineage-restricted precursor cells (such as a myocyte progenitor cell),which in turn can differentiate into other types of precursor cellsfurther down the pathway (such as a myocyte precursor), and then to anend-stage differentiated cell, which plays a characteristic role in acertain tissue type, and can or cannot retain the capacity toproliferate further.

Edited Human Cells

Provided herein are methods for editing a R838H, R838C, or R838Smutation in a GUCY2D gene in a human cell. Provided herein are gRNAs forediting a R838H, R838C, or R838S mutation in a GUCY2D gene in a humancell.

These methods and/or gRNAs disclosed herein can be used to edit apopulation of human cells. A sufficient number of human cells within acell population can be edited and used to treat a patient. For example,95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, or 50% of the human cellswithin a cell population can be edited and used to treat a patient. Inother examples, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, or 0.5%of the human cells within a cell population can be edited and used totreat a patient. In various examples, the edited human cells can befirst selected and cultured to expand the number of edited cells beforeadministering them to a patient.

Induced Pluripotent Stem Cells

The genetically engineered human cells described herein can be inducedpluripotent stem cells (iPSCs). An advantage of using iPSCs is that thecells can be derived from the same subject to which the progenitor cellsare to be administered. That is, a somatic cell can be obtained from asubject, reprogrammed to an induced pluripotent stem cell, and thenre-differentiated into a progenitor cell to be administered to thesubject (e.g., autologous cells). Because the progenitors areessentially derived from an autologous source, the risk of engraftmentrejection or allergic response can be reduced compared to the use ofcells from another subject or group of subjects. In addition, the use ofiPSCs negates the need for cells obtained from an embryonic source.Thus, in one aspect, the stem cells used in the disclosed methods arenot embryonic stem cells.

Although differentiation is generally irreversible under physiologicalcontexts, several methods have been recently developed to reprogramsomatic cells to iPSCs. Exemplary methods are known to those of skill inthe art and are described briefly herein below.

The term “reprogramming” refers to a process that alters or reverses thedifferentiation state of a differentiated cell (e.g., a somatic cell).Stated another way, reprogramming refers to a process of driving thedifferentiation of a cell backwards to a more undifferentiated or moreprimitive type of cell. It should be noted that placing many primarycells in culture can lead to some loss of fully differentiatedcharacteristics. Thus, simply culturing such cells included in the termdifferentiated cells does not render these cells non-differentiatedcells (e.g., undifferentiated cells) or pluripotent cells. Thetransition of a differentiated cell to pluripotency requires areprogramming stimulus beyond the stimuli that lead to partial loss ofdifferentiated character in culture. Reprogrammed cells also have thecharacteristic of the capacity of extended passaging without loss ofgrowth potential, relative to primary cell parents, which generally havecapacity for only a limited number of divisions in culture.

The cell to be reprogrammed can be either partially or terminallydifferentiated prior to reprogramming. Reprogramming can encompassecomplete reversion of the differentiation state of a differentiated cell(e.g., a somatic cell) to a pluripotent state or a multipotent state.Reprogramming can encompasse complete or partial reversion of thedifferentiation state of a differentiated cell (e.g., a somatic cell) toan undifferentiated cell (e.g., an embryonic-like cell). Reprogrammingcan result in expression of particular genes by the cells, theexpression of which further contributes to reprogramming. In certainexamples described herein, reprogramming of a differentiated cell (e.g.,a somatic cell) can cause the differentiated cell to assume anundifferentiated state (e.g., is an undifferentiated cell). Theresulting cells are referred to as “reprogrammed cells,” or “inducedpluripotent stem cells (iPSCs or iPS cells).”

Reprogramming can involve alteration, e.g., reversal, of at least someof the heritable patterns of nucleic acid modification (e.g.,methylation), chromatin condensation, epigenetic changes, genomicimprinting, etc., that occur during cellular differentiation.Reprogramming is distinct from simply maintaining the existingundifferentiated state of a cell that is already pluripotent ormaintaining the existing less than fully differentiated state of a cellthat is already a multipotent cell (e.g., a myogenic stem cell).Reprogramming is also distinct from promoting the self-renewal orproliferation of cells that are already pluripotent or multipotent,although the compositions and methods described herein can also be ofuse for such purposes, in some examples.

Many methods are known in the art that can be used to generatepluripotent stem cells from somatic cells. Any such method thatreprograms a somatic cell to the pluripotent phenotype would beappropriate for use in the methods described herein.

Reprogramming methodologies for generating pluripotent cells usingdefined combinations of transcription factors have been described. Mousesomatic cells can be converted to ES cell-like cells with expandeddevelopmental potential by the direct transduction of Oct4, Sox2, Klf4,and c-Myc; see, e.g., Takahashi and Yamanaka, Cell 126(4): 663-76(2006). iPSCs resemble ES cells, as they restore thepluripotency-associated transcriptional circuitry and much of theepigenetic landscape. In addition, mouse iPSCs satisfy all the standardassays for pluripotency: specifically, in vitro differentiation intocell types of the three germ layers, teratoma formation, contribution tochimeras, germline transmission [see, e.g., Maherali and Hochedlinger,Cell Stem Cell. 3(6):595-605 (2008)], and tetraploid complementation.

Human iPSCs can be obtained using similar transduction methods, and thetranscription factor trio, OCT4, SOX2, and NANOG, has been establishedas the core set of transcription factors that govern pluripotency; see,e.g., Budniatzky and Gepstein, Stem Cells Transl Med. 3(4):448-57(2014); Barrett et al., Stem Cells Trans Med 3:1-6 sctm.2014-0121(2014); Focosi et al., Blood Cancer Journal 4: e211 (2014). Theproduction of iPSCs can be achieved by the introduction of nucleic acidsequences encoding stem cell-associated genes into an adult, somaticcell, historically using viral vectors.

iPSCs can be generated or derived from terminally differentiated somaticcells, as well as from adult stem cells, or somatic stem cells. That is,a non-pluripotent progenitor cell can be rendered pluripotent ormultipotent by reprogramming. In such instances, it cannot be necessaryto include as many reprogramming factors as required to reprogram aterminally differentiated cell. Further, reprogramming can be induced bythe non-viral introduction of reprogramming factors, e.g., byintroducing the proteins themselves, or by introducing nucleic acidsthat encode the reprogramming factors, or by introducing messenger RNAsthat upon translation produce the reprogramming factors (see e.g.,Warren et al., Cell Stem Cell, 7(5):618-30 (2010). Reprogramming can beachieved by introducing a combination of nucleic acids encoding stemcell-associated genes, including, for example, Oct-4 (also known asOct-3/4 or Pouf51), Sox1, Sox2, Sox3, Sox 15, Sox 18, NANOG, Klf1, Klf2,Klf4, Klf5, NR5A2, c-Myc, 1-Myc, n-Myc, Rem2, Tert, and LIN28.Reprogramming using the methods and compositions described herein canfurther comprise introducing one or more of Oct-3/4, a member of the Soxfamily, a member of the Klf family, and a member of the Myc family to asomatic cell. The methods and compositions described herein can furthercomprise introducing one or more of each of Oct-4, Sox2, Nanog, c-MYCand Klf4 for reprogramming. As noted above, the exact method used forreprogramming is not necessarily critical to the methods andcompositions described herein. However, where cells differentiated fromthe reprogrammed cells are to be used in, e.g., human therapy, in oneaspect the reprogramming is not effected by a method that alters thegenome. Thus, in such examples, reprogramming can be achieved, e.g.,without the use of viral or plasmid vectors.

The efficiency of reprogramming (i.e., the number of reprogrammed cells)derived from a population of starting cells can be enhanced by theaddition of various agents, e.g., small molecules, as shown by Shi etal., Cell-Stem Cell 2:525-528 (2008); Huangfu et al., NatureBiotechnology 26(7):795-797 (2008) and Marson et al., Cell-Stem Cell 3:132-135 (2008). Thus, an agent or combination of agents that enhance theefficiency or rate of induced pluripotent stem cell production can beused in the production of patient-specific or disease-specific iPSCs.Some non-limiting examples of agents that enhance reprogrammingefficiency include soluble Wnt, Wnt conditioned media, BIX-01294 (a G9ahistone methyltransferase), PD0325901 (a MEK inhibitor), DNAmethyltransferase inhibitors, histone deacetylase (HDAC) inhibitors,valproic acid, 5′-azacytidine, dexamethasone, suberoylanilide,hydroxamic acid (SAHA), vitamin C, and trichostatin (TSA), among others.

Other non-limiting examples of reprogramming enhancing agents include:Suberoylanilide Hydroxamic Acid (SAHA (e.g., MK0683, vorinostat) andother hydroxamic acids), BML-210, Depudecin (e.g., (−)-Depudecin), HCToxin, Nullscript(4-(1,3-Dioxo-1H,3H-benzo[de]isoquinolin-2-yl)-N-hydroxybutanamide),Phenylbutyrate (e.g., sodium phenylbutyrate) and Valproic Acid ((VP A)and other short chain fatty acids), Scriptaid, Suramin Sodium,Trichostatin A (TSA), APHA Compound 8, Apicidin, Sodium Butyrate,pivaloyloxymethyl butyrate (Pivanex, AN-9), Trapoxin B, Chlamydocin,Depsipeptide (also known as FR901228 or FK228), benzamides (e.g., CI-994(e.g., N-acetyl dinaline) and MS-27-275), MGCD0103, NVP-LAQ-824, CBHA(m-carboxycinnaminic acid bishydroxamic acid), JNJ16241199, Tubacin,A-161906, proxamide, oxamflatin, 3-C1-UCHA (e.g.,6-(3-chlorophenylureido)caproic hydroxamic acid), AOE (2-amino-8-oxo-9,10-epoxydecanoic acid), CHAP31 and CHAP 50. Other reprogrammingenhancing agents include, for example, dominant negative forms of theHDACs (e.g., catalytically inactive forms), siRNA inhibitors of theHDACs, and antibodies that specifically bind to the HDACs. Suchinhibitors are available, e.g., from BIOMOL International, Fukasawa,Merck Biosciences, Novartis, Gloucester Pharmaceuticals, TitanPharmaceuticals, MethylGene, and Sigma Aldrich.

To confirm the induction of pluripotent stem cells for use with themethods described herein, isolated clones can be tested for theexpression of a stem cell marker. Such expression in a cell derived froma somatic cell identifies the cells as induced pluripotent stem cells.Stem cell markers can be selected from the non-limiting group includingSSEA3, SSEA4, CD9, Nanog, Fbxl5, Ecatl, Esgl, Eras, Gdf3, Fgf4, Cripto,Daxl, Zpf296, Slc2a3, Rexl, Utfl, and Natl. In one case, for example, acell that expresses Oct4 or Nanog is identified as pluripotent. Methodsfor detecting the expression of such markers can include, for example,RT-PCR and immunological methods that detect the presence of the encodedpolypeptides, such as Western blots or flow cytometric analyses.Detection can involve not only RT-PCR, but can also include detection ofprotein markers. Intracellular markers can be best identified viaRT-PCR, or protein detection methods such as immunocytochemistry, whilecell surface markers are readily identified, e.g., byimmunocytochemistry.

The pluripotent stem cell character of isolated cells can be confirmedby tests evaluating the ability of the iPSCs to differentiate into cellsof each of the three germ layers. As one example, teratoma formation innude mice can be used to evaluate the pluripotent character of theisolated clones. The cells can be introduced into nude mice andhistology and/or immunohistochemistry can be performed on a tumorarising from the cells. The growth of a tumor comprising cells from allthree germ layers, for example, further indicates that the cells arepluripotent stem cells.

Retinal Progenitor Cells and Photoreceptor Cells

In some examples, the genetically engineered human cells describedherein are photoreceptor cells or retinal progenitor cells (RPCs). RPCsare multipotent progenitor cells that can give rise to all six neuronsof the retina as well as the Müller glia. Müller glia are a type ofretinal glial cells and are the major glial component of the retina.Their function is to support the neurons of the retina and to maintainretinal homeostasis and integrity. Müller glia isolated from adult humanretinas have been shown to differentiate into rod photoreceptors.Functional characterization of such Müller glia-derived photoreceptorsby patch-clamp recordings has revealed that their electrical propertiesare comparable to those of adult rods (Giannelli et al., 2011, StemCells, (2):344-56). RPCs are gradually specified into lineage-restrictedprecursor cells during retinogenesis, which then maturate into theterminally differentiated neurons or Müuller glia. Fetal-derived humanretinal progenitor cells (hRPCs) exhibit molecular characteristicsindicative of a retinal progenitor state up to the sixth passage. Theydemonstrate a gradual decrease in the percentages of KI67-, SOX2-, andvimentin-positive cells from passages 1 to 6, whereas a sustainedexpression of nestin and PAX6 is seen through passage 6. Microarrayanalysis of passage 1 hRPCs demonstrates the expression of early retinaldevelopmental genes: VIM (vimentin), KI67, NES (nestin), PAX6, SOX2,HESS, GNL3, OTX2, DACH1, SIX6, and CHX10 (VSX2). The hRPCs arefunctional in nature and respond to excitatory neurotransmitters(Schmitt et al., 2009, Investigative Ophthalmology and Visual Sciences.2009; 50(12):5901-8). The outermost region of the retina contains asupportive retinal pigment epithelium (RPE) layer, which maintainsphotoreceptor health by transporting nutrients and recycling shedphotoreceptor parts. The RPE is attached to Bruch's membrane, anextracellular matrix structure at the interface between the choroid andretina. On the other side of the RPE, moving inwards towards theinterior of the eye, there are three layers of neurons: lightsensing rodand cone photoreceptors, a middle layer of connecting neurons (amacrine,bipolar and horizontal cells) and the innermost layer of ganglion cells,which transmit signals originating in the photoreceptor layer throughthe optic nerve and into the brain. In some aspects, the geneticallyengineered human cells described herein are photoreceptor cells, whichare specialized types of neurons found in the retina. Photoreceptorsconvert light into signals that are able to stimulate biologicalprocesses and are responsible for sight. Rods and cones are the twoclassic photoreceptor cells that contribute information to the visualsystem.

Isolating a Retinal Progenitor Cell and Photoreceptor Cell

Retinal cells, including progenitor cells may be isolated according toany method known in the art. For example, human retinal cells areisolated from fresh surgical specimens. The retinal pigment epithelium(RPE) is separated from the choroid by digesting the tissue with type IVcollagenase and the retinal pigment epithelium patches are cultured.Following the growth of 100-500 cells from the explant, the primarycultures are passaged (Ishida M. et al., Current Eye Research 1998;17(4):392-402) and characterized for expression of RPE markers. Rods areisolated by disruption of the biopsied retina using papain. Precautionsare taken to avoid a harsh disruption and improve cell yield. Theisolated cells are sorted to yield a population of pure rod cells andcharacterized further by immunostaining (Feodorova et al., MethodsX2015; 2:39-46).

In order to isolate cones, neural retina is identified, cut-out, andplaced on 10% gelatin. The inner retinal layers are isolated using alaser. The isolated cone monolayers are cultured for 18 hours andcompared with untreated retinas by light microscopy and transmissionmicroscopy to check for any structural damage. The cells arecharacterized for expression of cone-specific markers (Salchow et al.,Current Eye Research 2001; 22).

In order to isolate retinal progenitor cells, the biopsied retina isminced with dual scalpels and digested enzymatically in an incubator at37° C. The supernatants of the digested cells are centrifuged and thecells are resuspended in cell-free retinal progenitor-conditionedmedium. The cells are transferred to fibronectin-coated tissue cultureflasks containing fresh media and cultured (Klassen et al., Jornal ofNeuroscience Research 2004; 77:334-343).

Creating Patient Specific iPSCs

One step of the ex vivo methods of the present disclosure can involvecreating a patient-specific iPS cell, patient-specific iPS cells, or apatient specific iPS cell line. There are many established methods inthe art for creating patient specific iPS cells, as described inTakahashi and Yamanaka 2006; Takahashi, Tanabe et al. 2007. For example,the creating step can comprise: a) isolating a somatic cell, such as askin cell or fibroblast, from the patient; and b) introducing a set ofpluripotency-associated genes into the somatic cell in order to inducethe cell to become a pluripotent stem cell. The set ofpluripotency-associated genes can be one or more of the genes selectedfrom the group consisting of OCT4, SOX1, SOX2, SOX3, SOX15, SOX18,NANOG, KLF1, KLF2, KLF4, KLFS, c-MYC, n-MYC, REM2, TERT and LIN28.

Performing a Biopsy or Aspirate of the Patient's Bone Marrow

A biopsy or aspirate is a sample of tissue or fluid taken from the body.There are many different kinds of biopsies or aspirates. Nearly all ofthem involve using a sharp tool to remove a small amount of tissue. Ifthe biopsy will be on the skin or other sensitive area, numbing medicinecan be applied first. A biopsy or aspirate can be performed according toany of the known methods in the art. For example, in a bone marrowaspirate, a large needle is used to enter the pelvis bone to collectbone marrow.

Isolating a Mesenchymal Stem Cell

Mesenchymal stem cells can be isolated according to any method known inthe art, such as from a patient's bone marrow or peripheral blood. Forexample, marrow aspirate can be collected into a syringe with heparin.Cells can be washed and centrifuged on a Percoll™ density gradient.Cells, such as blood cells, liver cells, interstitial cells,macrophages, mast cells, and thymocytes, can be separated using densitygradient centrifugation media, Percoll™. The cells can then be culturedin Dulbecco's modified Eagle's medium (DMEM) (low glucose) containing10% fetal bovine serum (FBS) (Pittinger M F, Mackay A M, Beck S C etal., Science 1999; 284:143-147).

Differentiation of Genome-Edited iPSCs into Other Cell Types

Another step of the ex vivo methods of the present disclosure cancomprise differentiating the genome-edited iPSCs into photoreceptorcells. The differentiating step may be performed according to any methodknown in the art. For example, iPSCs can be used to generate retinalorganioids and photoreceptors as decribed in the art (Phillips et al.,Stem Cells, June 2014, 32(6): pgs. 1480-1492; Zhong et al. Nat. Commun.,2014, 5: pg 4047; Tucker et al., PLoS One, April 2011, 6(4): e18992).For example, hiPSC are differentiated into retinal progenitor cellsusing various treatments, including Wnt, Nodal, and Notch pathwayinhibitors (Noggin, Dk1, LeftyA, and DAPT) and other growth factors. Theretinal progenitor cells are further differentiated into photoreceptorcells, the treatment including: exposure to native retinal cells incoculture systems, RX+ or Mitf+ by subsequent treatment with retinoicacid and taurine, or exposure to several exogenous factors includingNoggin, Dkk1, DAPT, and insulin-like growth factor (Yang et al., StemCells International 2016).

Differentiation of Genome-Edited Mesenchymal Stem Cells intoPhotoreceptor Cells or Retinal Progenitor Cells

Another step of the ex vivo methods of the present disclosure cancomprise differentiating the genome-edited mesenchymal stem cells intophotoreceptor cells or retinal progenitor cells. The differentiatingstep can be performed according to any method known in the art.

Implanting Cells into Patients

Another step of the ex vivo methods of the present disclosure cancomprise implanting the photoreceptor cells or retinal progenitor cellsinto patients. This implanting step can be accomplished using any methodof implantation known in the art. For example, the genetically modifiedcells can be injected directly in the patient's blood or otherwiseadministered to the patient.

Another step of the ex vivo methods of the invention involves implantingthe photoreceptor cells or retinal progenitor cells into patients. Thisimplanting step can be accomplished using any method of implantationknown in the art. For example, the genetically modified cells can beinjected directly in the patient's eye or otherwise administered to thepatient.

Genetically Modified Cells

The term “genetically modified cell” refers to a cell that comprises atleast one genetic modification introduced by genome editing (e.g., usingthe CRISPR/Cas9/Cpf1 system). In some ex vivo examples herein, thegenetically modified cell can be genetically modified progenitor cell.In some in vivo examples herein, the genetically modified cell can be agenetically modified photoreceptor cell or retinal progenitor cell. Agenetically modified cell comprising an exogenous genome-targetingnucleic acid and/or an exogenous nucleic acid encoding agenome-targeting nucleic acid is contemplated herein.

The term “control treated population” describes a population of cellsthat has been treated with identical media, viral induction, nucleicacid sequences, temperature, confluency, flask size, pH, etc., with theexception of the addition of the genome editing components. Any methodknown in the art can be used to measure restoration of GUCY2D gene orprotein expression or activity, for example Western Blot analysis of theRetGC1 protein or real time PCR for quantifying GUCY2D mRNA.

The term “isolated cell” refers to a cell that has been removed from anorganism in which it was originally found, or a descendant of such acell. Optionally, the cell can be cultured in vitro, e.g., under definedconditions or in the presence of other cells. Optionally, the cell canbe later introduced into a second organism or re-introduced into theorganism from which it (or the cell from which it is descended) wasisolated.

The term “isolated population” with respect to an isolated population ofcells refers to a population of cells that has been removed andseparated from a mixed or heterogeneous population of cells. In somecases, the isolated population can be a substantially pure population ofcells, as compared to the heterogeneous population from which the cellswere isolated or enriched. In some cases, the isolated population can bean isolated population of human progenitor cells, e.g., a substantiallypure population of human progenitor cells, as compared to aheterogeneous population of cells comprising human progenitor cells andcells from which the human progenitor cells were derived.

The term “substantially enhanced,” with respect to a particular cellpopulation, refers to a population of cells in which the occurrence of aparticular type of cell is increased relative to pre-existing orreference levels, by at least 2-fold, at least 3-, at least 4-, at least5-, at least 6-, at least 7-, at least 8-, at least 9, at least 10-, atleast 20-, at least 50-, at least 100-, at least 400-, at least 1000-,at least 5000-, at least 20000-, at least 100000- or more folddepending, e.g., on the desired levels of such cells for amelioratingautosomal dominant CORD.

The term “substantially enriched” with respect to a particular cellpopulation, refers to a population of cells that is at least about 10%,about 20%, about 30%, about 40%, about 50%, about 60%, about 70% or morewith respect to the cells making up a total cell population.

The terms “substantially pure” with respect to a particular cellpopulation, refers to a population of cells that is at least about 75%,at least about 85%, at least about 90%, or at least about 95% pure, withrespect to the cells making up a total cell population. That is, theterms “substantially pure” or “essentially purified,” with regard to apopulation of progenitor cells, refers to a population of cells thatcontain fewer than about 20%, about 15%, about 10%, about 9%, about 8%,about 7%, about 6%, about 5%, about 4%, about 3%, about 2%, about 1%, orless than 1%, of cells that are not progenitor cells as defined by theterms herein.

Delivery

Guide RNA polynucleotides (RNA or DNA) and/or endonucleasepolynucleotide(s) (RNA or DNA) can be delivered by viral or non-viraldelivery vehicles known in the art. Alternatively, endonucleasepolypeptide(s) can be delivered by viral or non-viral delivery vehiclesknown in the art, such as electroporation or lipid nanoparticles. Infurther alternative aspects, the DNA endonuclease can be delivered asone or more polypeptides, either alone or pre-complexed with one or moreguide RNAs, or one or more crRNA together with a tracrRNA.

Polynucleotides can be delivered by non-viral delivery vehiclesincluding, but not limited to, nanoparticles, liposomes,ribonucleoproteins, positively charged peptides, small moleculeRNA-conjugates, aptamer-RNA chimeras, and RNA-fusion protein complexes.Some exemplary non-viral delivery vehicles are described in Peer andLieberman, Gene Therapy, 18: 1127-1133 (2011) (which focuses onnon-viral delivery vehicles for siRNA that are also useful for deliveryof other polynucleotides).

Polynucleotides, such as guide RNA, sgRNA, and mRNA encoding anendonuclease, can be delivered to a cell or a patient by a lipidnanoparticle (LNP).

A LNP refers to any particle having a diameter of less than 1000 nm, 500nm, 250 nm, 200 nm, 150 nm, 100 nm, 75 nm, 50 nm, or 25 nm.Alternatively, a nanoparticle can range in size from 1-1000 nm, 1-500nm, 1-250 nm, 25-200 nm, 25-100 nm, 35-75 nm, or 25-60 nm.

LNPs can be made from cationic, anionic, or neutral lipids. Neutrallipids, such as the fusogenic phospholipid DOPE or the membranecomponent cholesterol, can be included in LNPs as ‘helper lipids’ toenhance transfection activity and nanoparticle stability. Limitations ofcationic lipids include low efficacy owing to poor stability and rapidclearance, as well as the generation of inflammatory oranti-inflammatory responses.

LNPs can also be comprised of hydrophobic lipids, hydrophilic lipids, orboth hydrophobic and hydrophilic lipids.

Any lipid or combination of lipids that are known in the art can be usedto produce a LNP. Examples of lipids used to produce LNPs are: DOTMA,DOSPA, DOTAP, DMRIE, DC-cholesterol, DOTAP-cholesterol,GAP-DMORIE-DPyPE, and GL67A-DOPE-DMPE-polyethylene glycol (PEG).Examples of cationic lipids are: 98N12-5, C12-200, DLin-KC2-DMA (KC2),DLin-MC3-DMA (MC3), XTC, MD1, and 7C1. Examples of neutral lipids are:DPSC, DPPC, POPC, DOPE, and SM. Examples of PEG-modified lipids are:PEG-DMG, PEG-CerC14, and PEG-CerC20.

The lipids can be combined in any number of molar ratios to produce aLNP. In addition, the polynucleotide(s) can be combined with lipid(s) ina wide range of molar ratios to produce a LNP.

As stated previously, the site-directed polypeptide and genome-targetingnucleic acid can each be administered separately to a cell or a patient.On the other hand, the site-directed polypeptide can be pre-complexedwith one or more guide RNAs, or one or more crRNA together with atracrRNA. The pre-complexed material can then be administered to a cellor a patient. Such pre-complexed material is known as aribonucleoprotein particle (RNP).

RNA is capable of forming specific interactions with RNA or DNA. Whilethis property is exploited in many biological processes, it also comeswith the risk of promiscuous interactions in a nucleic acid-richcellular environment. One solution to this problem is the formation ofribonucleoprotein particles (RNPs), in which the RNA is pre-complexedwith an endonuclease. Another benefit of the RNP is protection of theRNA from degradation.

The endonuclease in the RNP can be modified or unmodified. Likewise, thegRNA, crRNA, tracrRNA, or sgRNA can be modified or unmodified. Numerousmodifications are known in the art and can be used.

The endonuclease and sgRNA can be generally combined in a 1:1 molarratio. Alternatively, the endonuclease, crRNA and tracrRNA can begenerally combined in a 1:1:1 molar ratio. However, a wide range ofmolar ratios can be used to produce a RNP.

AAV (Adeno Associated Virus)

A recombinant adeno-associated virus (AAV) vector can be used fordelivery. Techniques to produce rAAV particles, in which an AAV genometo be packaged that includes the polynucleotide to be delivered, rep andcap genes, and helper virus functions are provided to a cell arestandard in the art. Production of rAAV typically requires that thefollowing components are present within a single cell (denoted herein asa packaging cell): a rAAV genome, AAV rep and cap genes separate from(i.e., not in) the rAAV genome, and helper virus functions. The AAV repand cap genes can be from any AAV serotype for which recombinant viruscan be derived, and can be from a different AAV serotype than the rAAVgenome ITRs, including, but not limited to, AAV serotypes describedherein. Production of pseudotyped rAAV is disclosed in, for example,international patent application publication number WO 01/83692.

AAV sequences disclosed herein can comprise sgRNAs that target one ormore of the R838H mutation within the GUCY2D gene, the R838C mutationwithin the GUCY2D gene, and the R838S mutation within the GUCY2D gene.For example, pSIA012 comprises an AAV sequence (SEQ ID NO: 5506) thatencodes a sgRNA that targets the R838H mutation within the GUCY2D gene(SEQ ID NO: 5464). SEQ ID NO: 5464 is SEQ ID NOs: 5285 (sgRNAprotospacer sequence) and 5267 (sgRNA backbone sequence). Anotherplasmid (SEQ ID NO: 5470) comprises an AAV sequence that encodes a sgRNAthat targets the R838H mutation within the GUCY2D gene (SEQ ID NO:5465). SEQ ID NO: 5465 is SEQ ID NOs: 5286 (sgRNA protospacer sequence)and 5267 (sgRNA backbone sequence). pSIA015 comprises an AAV sequence(SEQ ID NO: 5507) that encodes a sgRNA that targets either the R838Hmutation or the R838C mutation within the GUCY2D gene (SEQ ID NO: 5466).SEQ ID NO: 5466 is SEQ ID NOs: 5398 (sgRNA protospacer sequence) and5267 (sgRNA backbone sequence).

AAV Serotypes

AAV particles packaging polynucleotides encoding compositions of thepresent disclosure, e.g., endonucleases, donor sequences, or RNA guidemolecules, of the present disclosure can comprise or be derived from anynatural or recombinant AAV serotype. According to the presentdisclosure, the AAV particles can utilize or be based on a serotypeselected from any of the following serotypes, and variants thereofincluding but not limited to AAV1, AAV10, AAV106.1/hu.37, AAV11,AAV114.3/hu.40, AAV12, AAV127.2/hu.41, AAV127.5/hu.42, AAV128.1/hu.43,AAV128.3/hu.44, AAV130.4/hu.48, AAV145.1/hu.53, AAV145.5/hu.54,AAV145.6/hu.55, AAV16.12/hu.11, AAV16.3, AAV16.8/hu.10, AAV161.10/hu.60,AAV161.6/hu.61, AAV1-7/rh.48, AAV1-8/rh.49, AAV2, AAV2.5T,AAV2-15/rh.62, AAV223.1, AAV223.2, AAV223.4, AAV223.5, AAV223.6,AAV223.7, AAV2-3/rh.61, AAV24.1, AAV2-4/rh.50, AAV2-5/rh.51, AAV27.3,AAV29.3/bb.1, AAV29.5/bb.2, AAV2G9, AAV-2-pre-miRNA-101, AAV3,AAV3.1/hu.6, AAV3.1/hu.9, AAV3-11/rh.53, AAV3-3, AAV33.12/hu.17,AAV33.4/hu.15, AAV33.8/hu.16, AAV3-9/rh.52, AAV3a, AAV3b, AAV4,AAV4-19/rh.55, AAV42.12, AAV42-10, AAV42-11, AAV42-12, AAV42-13,AAV42-15, AAV42-1b, AAV42-2, AAV42-3a, AAV42-3b, AAV42-4, AAV42-5a,AAV42-5b, AAV42-6b, AAV42-8, AAV42-aa, AAV43-1, AAV43-12, AAV43-20,AAV43-21, AAV43-23, AAV43-25, AAV43-5, AAV4-4, AAV44.1, AAV44.2,AAV44.5, AAV46.2/hu.28, AAV46.6/hu.29, AAV4-8/r11.64, AAV4-8/rh.64,AAV4-9/rh.54, AAV5, AAV52.1/hu.20, AAV52/hu.19, AAV5-22/rh.58,AAV5-3/rh.57, AAV54.1/hu.21, AAV54.2/hu.22, AAV54.4R/hu.27,AAV54.5/hu.23, AAV54.7/hu.24, AAV58.2/hu.25, AAV6, AAV6.1, AAV6.1.2,AAV6.2, AAV7, AAV7.2, AAV7.3/hu.7, AAV8, AAV-8b, AAV-8h, AAV9, AAV9.11,AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV9.84,AAV9.9, AAVA3.3, AAVA3.4, AAVA3.5, AAVA3.7, AAV-b, AAVC1, AAVC2, AAVC5,AAVCh.5, AAVCh.5R1, AAVcy.2, AAVcy.3, AAVcy.4, AAVcy.5, AAVCy.5R1,AAVCy.5R2, AAVCy.5R3, AAVCy.5R4, AAVcy.6, AAV-DJ, AAV-DJ8, AAVF3, AAVFS,AAV-h, AAVH-1/hu.1, AAVH2, AAVH-5/hu.3, AAVH6, AAVhE1.1, AAVhER1.14,AAVhEr1.16, AAVhEr1.18, AAVhER1.23, AAVhEr1.35, AAVhEr1.36, AAVhEr1.5,AAVhEr1.7, AAVhEr1.8, AAVhEr2.16, AAVhEr2.29, AAVhEr2.30, AAVhEr2.31,AAVhEr2.36, AAVhEr2.4, AAVhEr3.1, AAVhu.1, AAVhu.10, AAVhu.11, AAVhu.11,AAVhu.12, AAVhu.13, AAVhu.14/9, AAVhu.15, AAVhu.16, AAVhu.17, AAVhu.18,AAVhu.19, AAVhu.2, AAVhu.20, AAVhu.21, AAVhu.22, AAVhu.23.2, AAVhu.24,AAVhu.25, AAVhu.27, AAVhu.28, AAVhu.29, AAVhu.29R, AAVhu.3, AAVhu.31,AAVhu.32, AAVhu.34, AAVhu.35, AAVhu.37, AAVhu.39, AAVhu.4, AAVhu.40,AAVhu.41, AAVhu.42, AAVhu.43, AAVhu.44, AAVhu.44R1, AAVhu.44R2,AAVhu.44R3, AAVhu.45, AAVhu.46, AAVhu.47, AAVhu.48, AAVhu.48R1,AAVhu.48R2, AAVhu.48R3, AAVhu.49, AAVhu.5, AAVhu.51, AAVhu.52, AAVhu.53,AAVhu.54, AAVhu.55, AAVhu.56, AAVhu.57, AAVhu.58, AAVhu.6, AAVhu.60,AAVhu.61, AAVhu.63, AAVhu.64, AAVhu.66, AAVhu.67, AAVhu.7, AAVhu.8,AAVhu.9, AAVhu.t 19, AAVLG-10/rh.40, AAVLG-4/rh.38, AAVLG-9/hu.39,AAVLG-9/hu.39, AAV-LK01, AAV-LK02, AAVLK03, AAV-LK03, AAV-LK04,AAV-LK05, AAV-LK06, AAV-LK07, AAV-LK08, AAV-LK09, AAV-LK10, AAV-LK11,AAV-LK12, AAV-LK13, AAV-LK14, AAV-LK15, AAV-LK17, AAV-LK18, AAV-LK19,AAVN721-8/rh.43, AAV-PAEC, AAV-PAEC11, AAV-PAEC12, AAV-PAEC2, AAV-PAEC4,AAV-PAEC6, AAV-PAEC7, AAV-PAEC8, AAVpi.1, AAVpi.2, AAVpi.3, AAVrh.10,AAVrh.12, AAVrh.13, AAVrh.13R, AAVrh.14, AAVrh.17, AAVrh.18, AAVrh.19,AAVrh.2, AAVrh.20, AAVrh.21, AAVrh.22, AAVrh.23, AAVrh.24, AAVrh.25,AAVrh.2R, AAVrh.31, AAVrh.32, AAVrh.33, AAVrh.34, AAVrh.35, AAVrh.36,AAVrh.37, AAVrh.37R2, AAVrh.38, AAVrh.39, AAVrh.40, AAVrh.43, AAVrh.44,AAVrh.45, AAVrh.46, AAVrh.47, AAVrh.48, AAVrh.48, AAVrh.48.1,AAVrh.48.1.2, AAVrh.48.2, AAVrh.49, AAVrh.50, AAVrh.51, AAVrh.52,AAVrh.53, AAVrh.54, AAVrh.55, AAVrh.56, AAVrh.57, AAVrh.58, AAVrh.59,AAVrh.60, AAVrh.61, AAVrh.62, AAVrh.64, AAVrh.64R1, AAVrh.64R2,AAVrh.65, AAVrh.67, AAVrh.68, AAVrh.69, AAVrh.70, AAVrh.72, AAVrh.73,AAVrh.74, AAVrh.8, AAVrh.8R, AAVrh8R, AAVrh8R A586R mutant, AAVrh8RR533A mutant, BAAV, BNP61 AAV, BNP62 AAV, BNP63 AAV, bovine AAV, caprineAAV, Japanese AAV 10, true type AAV (ttAAV), UPENN AAV 10, AAV-LK16,AAAV, AAV Shuffle 100-1, AAV Shuffle 100-2, AAV Shuffle 100-3, AAVShuffle 100-7, AAV Shuffle 10-2, AAV Shuffle 10-6, AAV Shuffle 10-8, AAVSM 100-10, AAV SM 100-3, AAV SM 10-1, AAV SM 10-2, and/or AAV SM 10-8.

In some examples, the AAV serotype can be, or have, a mutation in theAAV9 sequence as described by N Pulicherla et al. (Molecular Therapy19(6):1070-1078 (2011), such as but not limited to, AAV9.9, AAV9.11,AAV9.13, AAV9.16, AAV9.24, AAV9.45, AAV9.47, AAV9.61, AAV9.68, AAV9.84.

In some examples, the AAV serotype can be, or have, a sequence asdescribed in U.S. Pat. No. 6,156,303, such as, but not limited to, AAV3B(SEQ ID NO: 1 and 10 of U.S. Pat. No. 6,156,303), AAV6 (SEQ ID NO: 2, 7and 11 of U.S. Pat. No. 6,156,303), AAV2 (SEQ ID NO: 3 and 8 of U.S.Pat. No. 6,156,303), AAV3A (SEQ ID NO: 4 and 9, of U.S. Pat. No.6,156,303), or derivatives thereof.

In some examples, the serotype can be AAVDJ or a variant thereof, suchas AAVDJ8 (or AAV-DJ8), as described by Grimm et al. (Journal ofVirology 82(12): 5887-5911 (2008)). The amino acid sequence of AAVDJ8can comprise two or more mutations in order to remove the heparinbinding domain (HBD). As a non-limiting example, the AAV-DJ sequencedescribed as SEQ ID NO: 1 in U.S. Pat. No. 7,588,772, can comprise twomutations: (1) R587Q where arginine (R; Arg) at amino acid 587 ischanged to glutamine (Q; Gln) and (2) R590T where arginine (R; Arg) atamino acid 590 is changed to threonine (T; Thr). As another non-limitingexample, can comprise three mutations: (1) K406R where lysine (K; Lys)at amino acid 406 is changed to arginine (R; Arg), (2) R587Q wherearginine (R; Arg) at amino acid 587 is changed to glutamine (Q; Gln) and(3) R590T where arginine (R; Arg) at amino acid 590 is changed tothreonine (T; Thr).

In some examples, the AAV serotype can be, or have, a sequence asdescribed in International Publication No. WO2015121501, such as, butnot limited to, true type AAV (ttAAV) (SEQ ID NO: 2 of WO2015121501),“UPenn AAV10” (SEQ ID NO: 8 of WO2015121501), “Japanese AAV10” (SEQ IDNO: 9 of WO2015121501), or variants thereof.

According to the present disclosure, AAV capsid serotype selection oruse can be from a variety of species. In one example, the AAV can be anavian AAV (AAAV). The AAAV serotype can be, or have, a sequence asdescribed in U.S. Pat. No. 9,238,800, such as, but not limited to, AAAV(SEQ ID NO: 1, 2, 4, 6, 8, 10, 12, and 14 of U.S. Pat. No. 9,238,800),or variants thereof.

In one example, the AAV can be a bovine AAV (BAAV). The BAAV serotypecan be, or have, a sequence as described in U.S. Pat. No. 9,193,769,such as, but not limited to, BAAV (SEQ ID NO: 1 and 6 of U.S. Pat. No.9,193,769), or variants thereof. The BAAV serotype can be or have asequence as described in U.S. Pat. No. 7,427,396, such as, but notlimited to, BAAV (SEQ ID NO: 5 and 6 of U.S. Pat. No. 7,427,396), orvariants thereof.

In one example, the AAV can be a caprine AAV. The caprine AAV serotypecan be, or have, a sequence as described in U.S. Pat. No. 7,427,396,such as, but not limited to, caprine AAV (SEQ ID NO: 3 of U.S. Pat. No.7,427,396), or variants thereof.

In other examples the AAV can be engineered as a hybrid AAV from two ormore parental serotypes. In one example, the AAV can be AAV2G9 whichcomprises sequences from AAV2 and AAV9. The AAV2G9 AAV serotype can be,or have, a sequence as described in United States Patent Publication No.US20160017005.

In one example, the AAV can be a serotype generated by the AAV9 capsidlibrary with mutations in amino acids 390-627 (VP1 numbering) asdescribed by Pulicherla et al. (Molecular Therapy 19(6):1070-1078(2011). The serotype and corresponding nucleotide and amino acidsubstitutions can be, but is not limited to, AAV9.1 (G1594C; D532H),AAV6.2 (T1418A and T1436X; V473D and I479K), AAV9.3 (T1238A; F413Y),AAV9.4 (T1250C and A1617T; F417S), AAV9.5 (A1235G, A1314T, A1642G,C1760T; Q412R, T548A, A587V), AAV9.6 (T1231A; F411I), AAV9.9 (G1203A,G1785T; W595C), AAV9.10 (A1500G, T1676C; M559T), AAV9.11 (A1425T,A1702C, A1769T; T568P, Q590L), AAV9.13 (A1369C, A1720T; N457H, T574S),AAV9.14 (T1340A, T1362C, T1560C, G1713A; L447H), AAV9.16 (A1775T;Q592L), AAV9.24 (T1507C, T1521G; W503R), AAV9.26 (A1337G, A1769C; Y446C,Q590P), AAV9.33 (A1667C; D556A), AAV9.34 (A1534G, C1794T; N512D),AAV9.35 (A1289T, T1450A, C1494T, A1515T, C1794A, G1816A; Q430L, Y484N,N98K, V6061), AAV9.40 (A1694T, E565V), AAV9.41 (A1348T, T1362C; T450S),AAV9.44 (A1684C, A1701T, A1737G; N562H, K567N), AAV9.45 (A1492T, C1804T;N498Y, L602F), AAV9.46 (G1441C, T1525C, T1549G; G481R, W509R, L517V),9.47 (G1241A, G1358A, A1669G, C1745T; S414N, G453D, K557E, T582I),AAV9.48 (C1445T, A1736T; P482L, Q579L), AAV9.50 (A1638T, C1683T, T1805A;Q546H, L602H), AAV9.53 (G1301A, A1405C, C1664T, G1811T; R134Q, S469R,A555V, G604V), AAV9.54 (C1531A, T1609A; L511I, L537M), AAV9.55 (T1605A;F535L), AAV9.58 (C1475T, C1579A; T492I, H527N), AAV.59 (T1336C; Y446H),AAV9.61 (A1493T; N498I), AAV9.64 (C1531A, A1617T; L511I), AAV9.65(C1335T, T1530C, C1568A; A523D), AAV9.68 (C1510A; P504T), AAV9.80(G1441A, G481R), AAV9.83 (C1402A, A1500T; P468T, E500D), AAV9.87(T1464C, T1468C; S490P), AAV9.90 (A1196T; Y399F), AAV9.91 (T1316G,A1583T, C1782G, T1806C; L439R, K528I), AAV9.93 (A1273G, A1421G, A1638C,C1712T, G1732A, A1744T, A1832T; S425G, Q474R, Q546H, P571L, G578R,T582S, D611V), AAV9.94 (A1675T; M559L) and AAV9.95 (T1605A; F535L).

In one example, the AAV can be a serotype comprising at least one AAVcapsid CD8+ T-cell epitope. As a non-limiting example, the serotype canbe AAV1, AAV2 or AAV8.

In one example, the AAV can be a variant, such as PHP.A or PHP.B asdescribed in Deverman. 2016. Nature Biotechnology. 34(2): 204-209.

In one example, the AAV can be a serotype selected from any of thosefound in SEQ ID NOs: 4697-5265 and Table 5.

In one example, the AAV can be encoded by a sequence, fragment orvariant as described in SEQ ID NOs: 4697-5265 and Table 5.

A method of generating a packaging cell involves creating a cell linethat stably expresses all of the necessary components for AAV particleproduction. For example, a plasmid (or multiple plasmids) comprising arAAV genome lacking AAV rep and cap genes, AAV rep and cap genesseparate from the rAAV genome, and a selectable marker, such as aneomycin resistance gene, are integrated into the genome of a cell. AAVgenomes have been introduced into bacterial plasmids by procedures suchas GC tailing (Samulski et al., 1982, Proc. Natl. Acad. S6. USA,79:2077-2081), addition of synthetic linkers containing restrictionendonuclease cleavage sites (Laughlin et al., 1983, Gene, 23:65-73) orby direct, blunt-end ligation (Senapathy & Carter, 1984, J. Biol. Chem.,259:4661-4666). The packaging cell line can then be infected with ahelper virus, such as adenovirus. The advantages of this method are thatthe cells are selectable and are suitable for large-scale production ofrAAV. Other examples of suitable methods employ adenovirus orbaculovirus, rather than plasmids, to introduce rAAV genomes and/or repand cap genes into packaging cells.

General principles of rAAV production are reviewed in, for example,Carter, 1992, Current Opinions in Biotechnology, 1533-539; and Muzyczka,1992, Curr. Topics in Microbial. and Immunol., 158:97-129). Variousapproaches are described in Ratschin et al., Mol. Cell. Biol. 4:2072(1984); Hermonat et al., Proc. Natl. Acad. Sci. USA, 81:6466 (1984);Tratschin et al., Mol. Cell. Biol. 5:3251 (1985); McLaughlin et al., J.Virol., 62:1963 (1988); and Lebkowski et al., 1988 Mol. Cell. Biol.,7:349 (1988). Samulski et al. (1989, J. Virol., 63:3822-3828); U.S. Pat.No. 5,173,414; WO 95/13365 and corresponding U.S. Pat. No. 5,658,776; WO95/13392; WO 96/17947; PCT/US98/18600; WO 97/09441 (PCT/US96/14423); WO97/08298 (PCT/US96/13872); WO 97/21825 (PCT/US96/20777); WO 97/06243(PCT/FR96/01064); WO 99/11764; Perrin et al. (1995) Vaccine13:1244-1250; Paul et al. (1993) Human Gene Therapy 4:609-615; Clark etal. (1996) Gene Therapy 3:1124-1132; U.S. Pat. Nos. 5,786,211;5,871,982; and 6,258,595.

AAV vector serotypes can be matched to target cell types. For example,the following exemplary cell types can be transduced by the indicatedAAV serotypes among others. See Table 5.

TABLE 5 Tissue/Cell Types and Serotypes Tissue/Cell Type Serotype LiverAAV3, AA5, AAV8, AAV9 Skeletal muscle AAV1, AAV7, AAV6, AAV8, AAV9Central nervous AAV1, AAV4, AAV5, AAV8, AAV9 system RPE AAV5, AAV4,AAV2, AAV8, AAV9, AAVrh8r Photoreceptor cells AAV5, AA8, AAV9, AAVrh8RLung AAV9, AAV5 Heart AAV8 Pancreas AAV8 Kidney AAV2, AAV8

In addition to adeno-associated viral vectors, other viral vectors canbe used. Such viral vectors include, but are not limited to, lentivirus,alphavirus, enterovirus, pestivirus, baculovirus, herpesvirus, EpsteinBarr virus, papovavirusr, poxvirus, vaccinia virus, and herpes simplexvirus.

In some cases, Cas9 mRNA, sgRNA targeting one or two loci in GUCY2Dgene, and donor DNA can each be separately formulated into lipidnanoparticles, or are all co-formulated into one lipid nanoparticle.

In some cases, Cas9 mRNA can be formulated in a lipid nanoparticle,while sgRNA and donor DNA can be delivered in an AAV vector.

Options are available to deliver the Cas9 nuclease as a DNA plasmid, asmRNA or as a protein. The guide RNA can be expressed from the same DNA,or can also be delivered as an RNA. The RNA can be chemically modifiedto alter or improve its half-life, or decrease the likelihood or degreeof immune response. The endonuclease protein can be complexed with thegRNA prior to delivery. Viral vectors allow efficient delivery; splitversions of Cas9 and smaller orthologs of Cas9 can be packaged in AAV,as can donors for HDR. A range of non-viral delivery methods also existthat can deliver each of these components, or non-viral and viralmethods can be employed in tandem. For example, nanoparticles can beused to deliver the protein and guide RNA, while AAV can be used todeliver a donor DNA.

Self-Inactivating (SIN) CRISPR-Cas Systems

Disclosed herein are “self-inactivating” (SIN) CRISPR-Cas systems. TheSIN CRISPR-Cas system can comprise one or more segments. The SINCRISPR-Cas system can be an AAV system. The SIN CRISPR-Cas system can bean AAV5 system.

A first segment can comprise a nucleotide sequence that encodes one ormore polypeptide inducing site-directed mutagenesis (e.g. Cas9 or Cpf1).The first segment can further comprise a start codon, a stop codon, anda poly (A) termination site. Such a polypeptide can be Streptococcuspyogenes Cas9 (SpCas9), Staphylococcus aureus Cas9 (SaCas9), or anyvariants thereof. A nucleotide sequence functioning as a promoter can beoperably linked to the first segment. The promoter can be aspatially-restricted promoter, bidirectional promoter driving sgRNA inone direction and Cas9 in the opposite orientation, or an induciblepromoter. The spatially-restricted promoter can be selected from thegroup consisting of: any ubiquitous promoter, any tissue or cell typespecific promoter, a hepatocyte-specific promoter, a neuron-specificpromoter, an adipocyte-specific promoter, a cardiomyocyte-specificpromoter, a skeletal muscle-specific promoter, lung progenitor cellspecific promoter, a photoreceptor-specific promoter, and a retinalpigment epithelial (RPE) selective promoter. The promoter can be a sEF1αpromoter or GRK1 promoter.

A second segment can comprise a nucleotide sequence that encodes asgRNA. The sgRNA can comprise any of SEQ ID NOs: 5282-5313 (FIG. 2A),5398-5409, 5434-5443 (FIG. 2D) and 5464-5466. The sgRNAs can besubstantially complementary to a SIN site and a genomic target sequence.By “hybridizable” or “complementary” or “substantially complementary” itis meant that a nucleic acid (e.g. RNA) comprises a sequence ofnucleotides that enables it to non-covalently bind, e.g.: formWatson-Crick base pairs, “anneal”, or “hybridize,” to another nucleicacid in a sequence-specific, antiparallel manner (i.e., a nucleic acidspecifically binds to a complementary nucleic acid) under theappropriate in vitro and/or in vivo conditions of temperature andsolution ionic strength. As is known in the art, standard Watson-Crickbase-pairing includes: adenine (A) pairing with thymidine (T), adenine(A) pairing with uracil (U), and guanine (G) pairing with cytosine (C)[DNA, RNA]. In some examples, the sgRNAs may be fully complementary tothe nucleotide sequence of the SIN site except for in at least onelocation. In some examples, the sgRNAs may be fully complementary to thenucleotide sequence of the SIN site except for in at least twolocations.

One or more third segments can be located at a 5′ end of the firstsegment (upstream of the start codon and/or downstream of thetranscriptional start site) or at a 3′ end of a first segment (betweenthe stop codon and poly (A) termination site). The one or more thirdsegments can be located at the 5′ end of the first segment and the 3′end of a first segment. The third segment can be less than 100nucleotides in length. For example, the third segment can be 20-99,30-99, 40-99, 50-99, 60-99, 70-99, 80-99, and 90-99 nucleotides inlength. The third segment can be less than 50 nucleotides in length. Forexample, the third segment can be 20-49, 25-49, 30-49, 35-49, 40-49, and45-49 nucleotides in length.

The one or more third segments can comprise a self-inactivating (SIN)site. The SIN site or R838 target site, as used herein, is either (1) a20-50 nucleotide sequence of the GUCY2D gene comprising the R838Hmutation (SEQ ID NO: 5478, 5479, and 5480), (2) a 20-50 nucleotidesequence of the GUCY2D gene comprising the R838C mutation (SEQ ID NOs:5481, 5482, and 5483), (3) a 20-50 nucleotide sequence of the GUCY2Dgene comprising the R838S mutation (SEQ ID NOs: 5484, 5485, and 5486),(4) a 20-50 nucleotide sequence of the GUCY2D gene comprising acombination of the R838C mutation and R838H mutation (SEQ ID NOs: 5490,5491, and 5492), or (5) a 20-50 nucleotide sequence of the GUCY2D genecomprising a combination of the R838S mutation and R838H mutation (SEQID NOs: 5487, 5488, and 5489) (Table 6). The SIN site comprisesprotospacer adjacent motifs (PAMs).

TABLE 6 SEQ ID SIN-AAV SpCas9 NO: 5′ SIN site sequence Allele(s)SIN-AAV SpCas9 ver. 1 5478 ggaggatctgatccgggagcacacggaggagctgga HSIN-AAV SpCas9 ver. 1 5481 ggaggatctgatccgggagtgcacggaggagctgga CSIN-AAV SpCas9 ver. 1 5484 ggaggatctgatccgggagagcacggaggagctgga SSIN-AAV SpCas9 ver. 1 5487 ggaggatctgatccgggagaacacggaggagctgga SHSIN-AAV SpCas9 ver. 1 5490 ggaggatctgatccgggagtacacggaggagctgga CHSIN-AAV SpCas9 ver. 2 5479 aggatctgatccgggagcacacggaggagctgga HSIN-AAV SpCas9 ver. 2 5482 aggatctgatccgggagtgcacggaggagctgga CSIN-AAV SpCas9 ver. 2 5485 aggatctgatccgggagagcacggaggagctgga SSIN-AAV SpCas9 ver. 2 5488 aggatctgatccgggagaacacggaggagctgga SHSIN-AAV SpCas9 ver. 2 5491 aggatctgatccgggagtacacggaggagctgga CH SEQ IDSIN-AAV SpCas9 NO: 3′ SIN site sequence Allele(s)SIN-AAV SpCas9 ver. 1 & 2 5480 tccagctcctccgtgtgctcccggatcagatcctcc HSIN-AAV SpCas9 ver. 1 & 2 5483 tccagctcctccgtgcactcccggatcagatcctcc CSIN-AAV SpCas9 ver. 1 & 2 5486 tccagctcctccgtgctctcccggatcagatcctcc SSIN-AAV SpCas9 ver. 1 & 2 5489 tccagctcctccgtgttctcccggatcagatcctcc SHSIN-AAV SpCas9 ver. 1 & 2 5492 tccagctcctccgtgtactcccggatcagatcctcc CH

The spacer sequence of a gRNA or sgRNA hybridizes to the strandcomplementary to the protospacer sequence located within the SIN site,which leads to editing by the gRNA-endonuclease complex or thesgRNA-endonuclease complex and eventually results in inactivation of theendonuclease (e.g. Cas9 or Cpf1). SIN sites that comprise a 20-50nucleotide sequence of the GUCY2D gene comprising the R838H mutation canbe targeted with any of the sgRNAs comprising SEQ ID NOs: 5282-5313,5398-5409, and 5434-5443 even though one or more of the sgRNAs may notbe fully complementary to the nucleotide sequence of the SIN site in atleast 1-2 locations. SIN sites that comprise a 20-50 nucleotide sequenceof the GUCY2D gene comprising the R838C mutation can be targeted withany of the sgRNAs comprising SEQ ID NOs: 5282-5313, 5398-5409, and5434-5443 even though one or more of these sgRNAs may not be fullycomplementary to the nucleotide sequence of the SIN site in at least 1-2locations. SIN sites that comprise a 20-50 nucleotide sequence of theGUCY2D gene comprising the R838S mutation can be targeted with any ofthe sgRNAs comprising SEQ ID NOs: 5282-5313, 5398-5409, and 5434-5443even though one or more of these sgRNAs may not be fully complementaryto the nucleotide sequence of the SIN site in at least 1-2 locations.SIN sites that comprise a 20-50 nucleotide sequence of the GUCY2D genecomprising a combination of both the R838C mutation and R838H mutationcan be targeted with any of the sgRNAs comprising SEQ ID NOs: 5282-5313,5398-5409, and 5434-5443 even though one or more of these sgRNAs may notbe fully complementary to the nucleotide sequence of the SIN site in atleast 1-2 locations. SIN sites that comprise a 20-50 nucleotide sequenceof the GUCY2D gene comprising a combination of both the R838S mutationand R838H mutation can be targeted with any of the sgRNAs comprising SEQID NOs: 5282-5313, 5398-5409, and 5434-5443 even though one or more ofthese sgRNAs may not be fully complementary to the nucleotide sequenceof the SIN site in at least 1-2 locations.

In other examples, the SIN site can be shorter in length compared to thesequences listed in Table 6. For example, the SIN site can be any one ofthe sequences in SEQ ID NOs: 5324-5355 (FIG. 2B) and a PAM. The SIN sitecan be any one of the sequences in 5410-5421 (FIG. 2E) and a PAM. TheSIN site can be any one of the sequences in 5444-5453 (FIG. 2E) and aPAM. The SIN site can be any one of the sequences in SEQ ID NOs:5366-5397 (FIG. 2C) and a PAM. The SIN site can be any one of thesequences in SEQ ID NOs: 5422-5433 (FIG. 2F) and a PAM. The SIN site canbe any one of the sequences in 5454-5463 (FIG. 2F) and a PAM.

In other examples, the SIN site can be shorter than the correspondingprotospacer sequence of the sgRNA. For example, a protospacer sequencefor a sgRNA may be 20 nucleotides in length whereas the correspondingSIN site may be shorter (only 19, 18, or 17 nucleotides in length) and aPAM. This shortened SIN site (that still corresponds to the spacersequence of the sgRNA) will allow the genomic target sequence to becleaved more efficiently than the shortened SIN site. For this reason,any one of the sequences in SEQ ID NOs: 5324-5355 (FIG. 2B), SEQ ID NOs:5410-5421 (FIG. 2E), SEQ ID NOs: 5444-5453 (FIG. 2E), SEQ ID NOs:5366-5397 (FIG. 2C), SEQ ID NOs: 5422-5433 (FIG. 2F), and SEQ ID NOs:5454-5463 (FIG. 2F) can be shortened by 1, 2, or 3 nucleotides and usedas a SIN site along with a PAM sequence. In these examples, the SINsites may be less than 20 nucleotides in length.

In the SIN-AAV system, the endonuclease can be guided by one or moresgRNAs to one or more genomic target sequences. The one or more genomictarget sequences can be a R838H mutation within the GUCY2D gene, a R838Cmutation within the GUCY2D gene, or a R838S mutation within the GUCY2Dgene. The endonuclease can be further guided to the SIN-AAV system thatis expressing the endonuclease and the system's components. Examples ofSIN-AAV system components that can be targeted include: essentialsequences of a vector of the SIN-AAV system (e.g. viral invertedterminal repeats), promoters driving expression of genes important forediting (e.g. sgRNA or endonuclease genes), the open reading frame (ORF)of Cas9 or Cpf1, introns of encoded genes, or non-coding regions located5′ or 3′ of the Cas9 or Cpf1 ORF (SIN sites). This leads toself-limiting editing activity which results in editing of one or moretarget genomic loci, and, thereafter, reduced or eliminated expressionof the endonuclease and/or other essential components of the system(e.g. sgRNAs). This self-limited expression of genes in the SIN-AAVsystem can result in reduced off-target editing and reduced risk ofsuccessfully edited cells being targeted by the patient's immune system.

One or more vectors can encode the disclosed SIN-AAV systems. If onlyone vector encodes the entire SIN-AAV system, then the system isreferred to as an “all-in-one” SIN system. For example, the firstsegment, second segment, and third segment can be provided together inan “all-in-one” SIN AAV system. If two vectors encode the entire SIN-AAVsystem, then the system is referred to as an “all-in-two” SIN system.For example, the first segment and third segment can be provided in afirst vector and the second segment can be provided in a second vectorfor an “all-in-two” SIN AAV system.

All-in-One SIN-AAV Systems

In one example, an all-in-one SIN system can comprise a vectorcomprising an endonuclease ORF and a sgRNA gene. The vector can furthercomprise SIN sites at locations 5′ and 3′ of the endonuclease ORF. ThesgRNA can be substantially complementary to the SIN site. The sgRNA canalso be substantially complementary to a genomic target sequence. Thus,the sequence of the sgRNA is such that it can hybridize with both theSIN sites on the vector and with one or more genomic target sequences.When hybridizing with the one or more genomic targets or with the SINsites, the sgRNA may comprise one or more mismatched bases. The systemcan lead to self-limited editing at the targeted genomic loci, followedby excision and/or inactivation of the endonuclease gene.

In another example, an all-in-one SIN system can comprise a vectorcomprising an endonuclease ORF, a first sgRNA gene, and a second sgRNAgene. The vector can further comprise SIN sites at locations 5′ and 3′of the endonuclease ORF. The sequence of the first sgRNA is such that itcan hybridize with one or more genomic target sequences. The sequence ofthe second sgRNA is such that it can hybridize with the SIN sites on thevector. When hybridizing with the one or more genomic targets or withthe SIN sites, the sgRNAs can comprise one or more mismatched bases.Additional sgRNAs can be incorporated into the system to allow forediting of additional genomic or SIN system targets. The system can leadto self-limited editing at the targeted genomic loci, followed byexcision and/or inactivation of the endonuclease gene.

In another example, an all-in-one SIN system can comprise a vectorcomprising an endonuclease ORF, a first sgRNA gene, and a second sgRNAgene. The sequence of the first sgRNA is such that it can hybridize withone or more genomic target sequences. The sequence of the second sgRNAis such that it can hybridize within or near the endonuclease ORF (Cas9or Cpf1) on the vector, leading to inactivation of the endonuclease genevia indel generation. Additional sgRNAs can be incorporated into thesystem to allow for editing of additional genomic or SIN system targets.When hybridizing with the one or more genomic targets or theendonuclease ORF, the two or more sgRNAs may comprise one or moremismatched bases. The system can lead to self-limited editing at thetargeted genomic loci, followed by inactivation of the endonucleasegene.

In all-in-one systems such as those described above, production ofappropriate viral vectors can be challenging due to inactivation of theendonuclease gene that occurs earlier than desired and accumulation ofmutagenized SIN sites on DNA packaged in AAV capsids (e.g. duringproduction and packaging of the viral vector in a cell line of choice).To solve this problem, the endonuclease ORF and/or the sgRNA genes thatdirect editing at the endonuclease gene locus can be expressed from oneor more cell/tissue-specific promoters. The cell/tissue specificpromoters can be active in the cells where editing is desired andinactive earlier in the cells used for production and packaging of thevectors. In addition, one or more inducible promoter systems can be usedto control expression of genes of interest, such astetracycline-controlled transcriptional activation (i.e. tet-ON ortet-OFF). Other solutions to the premature inactivation problem includeregulating gene expression with miRNAs, small interfering RNAs, shorthairpin RNAs, other antisense oligonucleotides, blocking transcriptionof sgRNA (e.g. the use of a tet-OFF system), or inhibiting sgRNA loadingonto Cas9.

All-in-Two SIN-AAV Systems

In one example, an all-in-two SIN system can comprise a first vector toprovide an ORF encoding an endonuclease (FIG. 11A-B or 14A-B). SIN sitescan flank the endonuclease ORF at 5′ and 3′ locations on the firstvector (FIG. 11A-B or 14A-B). The SIN site can be any one of SEQ ID NOs:5478-5492, as shown in Table 6. The SIN site can be shorter in lengthcompared to the sequences listed in Table 6. For example, the SIN sitecan be any one of the sequences in SEQ ID NOs: 5324-5355 (FIG. 2B) and aPAM. The SIN site can be any one of the sequences in 5410-5421 (FIG. 2E)and a PAM. The SIN site can be any one of the sequences in 5444-5453(FIG. 2E) and a PAM. The SIN site can be any one of the sequences in SEQID NOs: 5366-5397 (FIG. 2C) and a PAM. The SIN site can be any one ofthe sequences in SEQ ID NOs: 5422-5433 (FIG. 2F) and a PAM. The SIN sitecan be any one of the sequences in 5454-5463 (FIG. 2F) and a PAM. Inother examples, the SIN site can be shorter than the correspondingprotospacer sequence of the sgRNA. For example, a protospacer sequencefor a sgRNA may be 20 nucleotides in length whereas the correspondingSIN site may be shorter (only 19, 18, or 17 nucleotides in length) and aPAM. This shortened SIN site (that still corresponds to the spacersequence of the sgRNA) will allow the genomic target sequence to becleaved more efficiently than the shortened SIN site. For this reason,any one of the sequences in SEQ ID NOs: 5324-5355 (FIG. 2B), SEQ ID NOs:5410-5421 (FIG. 2E), SEQ ID NOs: 5444-5453 (FIG. 2E), SEQ ID NOs:5366-5397 (FIG. 2C), SEQ ID NOs: 5422-5433 (FIG. 2F), and SEQ ID NOs:5454-5463 (FIG. 2F) can be shortened by 1, 2, or 3 nucleotides and usedas a SIN site along with a PAM sequence. In a second vector, a singlesgRNA can be encoded (FIG. 11D). The sgRNA can comprise any of SEQ IDNOs: 5282-5313 (FIG. 2A), 5398-5409 (FIG. 2D), 5434-5443 (FIG. 2D) and5464-5466. The sgRNA can be substantially complementary to the SIN site.The sgRNA can also be substantially complementary to a genomic targetsequence. Thus, the sequence of the sgRNA can be such that it canhybridize with both the SIN sites on the first vector and with one ormore genomic target sequences (e.g. the R838H mutation within GUCY2D,the R838C mutation within GUCY2D, or the R838S mutation within GUCY2D).When hybridizing with the one or more genomic targets or with the SINsites, the sgRNA may comprise one or more mismatched bases. The systemcan lead to self-limited editing at the targeted genomic loci, followedby excision and/or inactivation of the endonuclease gene.

In another example, an all-in-two SIN system can comprise a first vectorto provide an ORF encoding an endonuclease. SIN sites can flank theendonuclease ORF at 5′ and 3′ locations on the first vector. The SINsite can be any one of SEQ ID NOs: 5478-5492, as shown in Table 6. TheSIN site can be shorter in length compared to the sequences listed inTable 6. For example, the SIN site can be any one of the sequences inSEQ ID NOs: 5324-5355 (FIG. 2B) and a PAM. The SIN site can be any oneof the sequences in 5410-5421 (FIG. 2E) and a PAM. The SIN site can beany one of the sequences in 5444-5453 (FIG. 2E) and a PAM. The SIN sitecan be any one of the sequences in SEQ ID NOs: 5366-5397 (FIG. 2C) and aPAM. The SIN site can be any one of the sequences in SEQ ID NOs:5422-5433 (FIG. 2F) and a PAM. The SIN site can be any one of thesequences in 5454-5463 (FIG. 2F) and a PAM. In other examples, the SINsite can be shorter than the corresponding protospacer sequence of thesgRNA. For example, a protospacer sequence for a sgRNA may be 20nucleotides in length whereas the corresponding SIN site may be shorter(only 19, 18, or 17 nucleotides in length) and a PAM. This shortened SINsite (that still corresponds to the spacer sequence of the sgRNA) willallow the genomic target sequence to be cleaved more efficiently thanthe shortened SIN site. For this reason, any one of the sequences in SEQID NOs: 5324-5355 (FIG. 2B), SEQ ID NOs: 5410-5421 (FIG. 2E), SEQ IDNOs: 5444-5453 (FIG. 2E), SEQ ID NOs: 5366-5397 (FIG. 2C), SEQ ID NOs:5422-5433 (FIG. 2F), and SEQ ID NOs: 5454-5463 (FIG. 2F) can beshortened by 1, 2, or 3 nucleotides and used as a SIN site along with aPAM sequence. The all-in-two system can further comprise a secondvector, comprising two sgRNA genes. When expressed from the secondvector, a first sgRNA can bind with an endonuclease molecule and directediting at one or more genomic target loci (e.g. the R838H mutationwithin GUCY2D, the R838C mutation within GUCY2D, or the R838S mutationwithin GUCY2D). The first sgRNA can comprise any of SEQ ID NOs:5282-5313 (FIG. 2A), 5398-5409, 5434-5443 (FIG. 2D) and 5464-5466. Whenexpressed from the second vector, a second sgRNA can bind with anendonuclease molecule and direct editing at the SIN sites. AdditionalsgRNAs can be incorporated into the system to allow for editing ofadditional genomic or SIN system targets. When hybridizing with the oneor more genomic targets or with the SIN sites, the two or more sgRNAsmay comprise one or more mismatched bases. In some examples, the one ormore sgRNAs that target genomic loci may be encoded on the first vector,or a combination of both the first and second vectors. The system canlead to self-limited editing at the targeted genomic loci, followed byexcision and/or inactivation of the endonuclease gene.

In another example, an all-in-two SIN system can comprise a first vectorcomprising an endonuclease ORF, and a second vector comprising two sgRNAgenes. When expressed from the second vector, a first sgRNA can bindwith an endonuclease molecule and direct editing at one or more genomictarget loci (e.g. the R838H mutation within GUCY2D, the R838C mutationwithin GUCY2D, or the R838S mutation within GUCY2D). The first sgRNA cancomprise any of SEQ ID NOs: 5282-5313 (FIG. 2A), 5398-5409, 5434-5443(FIG. 2D) and 5464-5466. When expressed from the second vector, a secondsgRNA can bind with an endonuclease molecule and direct editing withinor near the endonuclease ORF (Cas9 or Cpf1) on the first vector, leadingto inactivation of the endonuclease gene via indel generation.Additional sgRNAs can be incorporated into the system to allow forediting of additional genomic or SIN system targets. When hybridizingwith the one or more genomic targets or within or near the endonucleaseORF, the two or more sgRNAs may comprise one or more mismatched bases.In some examples, the one or more sgRNAs that target genomic loci may beencoded on the first vector, or a combination of both the first andsecond vectors. The system can lead to self-limited editing at thetargeted genomic loci, followed by inactivation of the endonucleasegene.

Lentivirus

In some aspects, lentiviral vectors or particles can be used as deliveryvehicles. Lentiviruses (LV) are subgroup of the Retroviridae family ofviruses. Lentiviral particles are able to integrate their geneticmaterial into the genome of a target/host cell. Examples of lentivirusinclude the Human Immunodeficiency Viruses: HIV-1 and HIV-2, JembranaDisease Virus (JDV), equine infectious anemia virus (EIAV), equineinfectious anemia virus, visna-maedi and caprine arthritis encephalitisvirus (CAEV), the Simian Immunodeficiency Virus (SIV), felineimmunodeficiency virus (FIV), bovine immunodeficiency virus (BIV). LV'sare capable of infecting both dividing and non-dividing cells due totheir unique ability to pass through a target cell's intact nuclearmembrane Greenberg et al., University of Berkeley, Calif.; 2006).Lentiviral particles that form the gene delivery vehicle are replicationdefective and are generated by attenuating the HIV virulence genes. Forexample, the genes Vpu, Vpr, Nef, Env, and Tat are excised making thevector biologically safe. Lentiviral vehicles, for example, derived fromHIV-1/HIV-2 can mediate the efficient delivery, integration andlong-term expression of transgenes into non-dividing cells. As usedherein, the term “recombinant” refers to a vector or other nucleic acidcontaining both lentiviral sequences and non-lentiviral retroviralsequences.

In order to produce a lentivirus that is capable of infecting hostcells, three types of vectors need to be co-expressed in virus producingcells: a backbone vector containing the transgene of interests andself-inactivating 3′-LTR regions, one construct expressing viralstructure proteins, and one vector encoding vesicular stomatitis virusglycoprotein (VSVG) for encapsulation (Naldini, L. et al., Science 1996;272, 263-267). Separation of the Rev gene from other structural genesfurther increases the biosafety by reducing the possibility of reverserecombination. Cell lines that can be used to produce high-titerlentiviral particles may include, but are not limited to 293T cells,293FT cells, and 293SF-3F6 cells (Witting et al., Human Gene Therapy,2012; 23: 243-249; Ansorge et al., Joural of Genetic Medicne, 2009; 11:868-876).

Methods for generating recombinant lentiviral particles are discussed inthe art, for example, WO 2013076309 (PCT/EP2012/073645); WO 2009153563(PCT/GB2009/001527); U.S. Pat. Nos. 7,629,153; and 6,808,905.

Cell types such as photoreceptors, retinal pigment epithelium, andganglion cells have been successfully targeted with LV vector. Theefficiency of delivery to photoreceptors and ganglion cells issignificantly higher with AAV than LV vectors.

Pharmaceutically Acceptable Carriers

The ex vivo methods of administering progenitor cells to a subjectcontemplated herein involve the use of therapeutic compositionscomprising progenitor cells.

Therapeutic compositions can contain a physiologically tolerable carriertogether with the cell composition, and optionally at least oneadditional bioactive agent as described herein, dissolved or dispersedtherein as an active ingredient. In some cases, the therapeuticcomposition is not substantially immunogenic when administered to amammal or human patient for therapeutic purposes, unless so desired.

In general, the progenitor cells described herein can be administered asa suspension with a pharmaceutically acceptable carrier. One of skill inthe art will recognize that a pharmaceutically acceptable carrier to beused in a cell composition will not include buffers, compounds,cryopreservation agents, preservatives, or other agents in amounts thatsubstantially interfere with the viability of the cells to be deliveredto the subject. A formulation comprising cells can include e.g., osmoticbuffers that permit cell membrane integrity to be maintained, andoptionally, nutrients to maintain cell viability or enhance engraftmentupon administration. Such formulations and suspensions are known tothose of skill in the art and/or can be adapted for use with theprogenitor cells, as described herein, using routine experimentation.

A cell composition can also be emulsified or presented as a liposomecomposition, provided that the emulsification procedure does notadversely affect cell viability. The cells and any other activeingredient can be mixed with excipients that are pharmaceuticallyacceptable and compatible with the active ingredient, and in amountssuitable for use in the therapeutic methods described herein.

Additional agents included in a cell composition can includepharmaceutically acceptable salts of the components therein.Pharmaceutically acceptable salts include the acid addition salts(formed with the free amino groups of the polypeptide) that are formedwith inorganic acids, such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, tartaric, mandelic and the like.Salts formed with the free carboxyl groups can also be derived frominorganic bases, such as, for example, sodium, potassium, ammonium,calcium or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like.

Physiologically tolerable carriers are well known in the art. Exemplaryliquid carriers are sterile aqueous solutions that contain no materialsin addition to the active ingredients and water, or contain a buffersuch as sodium phosphate at physiological pH value, physiological salineor both, such as phosphate-buffered saline. Still further, aqueouscarriers can contain more than one buffer salt, as well as salts such assodium and potassium chlorides, dextrose, polyethylene glycol and othersolutes. Liquid compositions can also contain liquid phases in additionto and to the exclusion of water. Exemplary of such additional liquidphases are glycerin, vegetable oils such as cottonseed oil, andwater-oil emulsions. The amount of an active compound used in the cellcompositions that is effective in the treatment of a particular disorderor condition can depend on the nature of the disorder or condition, andcan be determined by standard clinical techniques.

Guide RNA Formulation

Guide RNAs of the present disclosure can be formulated withpharmaceutically acceptable excipients such as carriers, solvents,stabilizers, adjuvants, diluents, etc., depending upon the particularmode of administration and dosage form. Guide RNA compositions can beformulated to achieve a physiologically compatible pH, and range from apH of about 3 to a pH of about 11, about pH 3 to about pH 7, dependingon the formulation and route of administration. In some cases, the pHcan be adjusted to a range from about pH 5.0 to about pH 8. In somecases, the compositions can comprise a therapeutically effective amountof at least one compound as described herein, together with one or morepharmaceutically acceptable excipients. Optionally, the compositions cancomprise a combination of the compounds described herein, or can includea second active ingredient useful in the treatment or prevention ofbacterial growth (for example and without limitation, anti-bacterial oranti-microbial agents), or can include a combination of reagents of thepresent disclosure.

Suitable excipients include, for example, carrier molecules that includelarge, slowly metabolized macromolecules such as proteins,polysaccharides, polylactic acids, polyglycolic acids, polymeric aminoacids, amino acid copolymers, and inactive virus particles. Otherexemplary excipients can include antioxidants (for example and withoutlimitation, ascorbic acid), chelating agents (for example and withoutlimitation, EDTA), carbohydrates (for example and without limitation,dextrin, hydroxyalkylcellulose, and hydroxyalkylmethylcellulose),stearic acid, liquids (for example and without limitation, oils, water,saline, glycerol and ethanol), wetting or emulsifying agents, pHbuffering substances, and the like.

Administration & Efficacy

The terms “administering,” “introducing” and “transplanting” can be usedinterchangeably in the context of the placement of cells, e.g.,progenitor cells, into a subject, by a method or route that results inat least partial localization of the introduced cells at a desired site,such as a site of injury or repair, such that a desired effect(s) isproduced. The cells e.g., progenitor cells, or their differentiatedprogeny can be administered by any appropriate route that results indelivery to a desired location in the subject where at least a portionof the implanted cells or components of the cells remain viable. Theperiod of viability of the cells after administration to a subject canbe as short as a few hours, e.g., twenty-four hours, to a few days, toas long as several years, or even the life time of the patient, i.e.,long-term engraftment. For example, in some aspects described herein, aneffective amount of photoreceptor cells or retinal progenitor cells isadministered via a systemic route of administration, such as anintraperitoneal or intravenous route.

The terms “administering,” “introducing” and “transplanting” can also beused interchangeably in the context of the placement of at least one ofa gRNA, sgRNA, and an endonuclease into a subject, by a method or routethat results in at least partial localization of the introduced gRNA,sgRNA, and/or endonuclease at a desired site, such as a site of injuryor repair, such that a desired effect(s) is produced. The gRNA, sgRNA,and/or endonuclease can be administered by any appropriate route thatresults in delivery to a desired location in the subject.

The terms “individual,” “subject,” “host” and “patient” are usedinterchangeably herein and refer to any subject for whom diagnosis,treatment or therapy is desired. In some aspects, the subject is amammal. In some aspects, the subject is a human being.

When provided prophylactically, progenitor cells described herein can beadministered to a subject in advance of any symptom of autosomaldominant CORD. Accordingly, the prophylactic administration of aprogenitor cell population serves to prevent autosomal dominant CORD.

A progenitor cell population being administered according to the methodsdescribed herein can comprise allogeneic progenitor cells obtained fromone or more donors. Such progenitors can be of any cellular or tissueorigin, e.g., liver, muscle, cardiac, etc. “Allogeneic” refers to aprogenitor cell or biological samples comprising progenitor cellsobtained from one or more different donors of the same species, wherethe genes at one or more loci are not identical. For example, aphotoreceptor or retinal progenitor cell population being administeredto a subject can be derived from one more unrelated donor subjects, orfrom one or more non-identical siblings. In some cases, syngeneicprogenitor cell populations can be used, such as those obtained fromgenetically identical animals, or from identical twins. The progenitorcells can be autologous cells; that is, the progenitor cells areobtained or isolated from a subject and administered to the samesubject, i.e., the donor and recipient are the same.

The term “effective amount” refers to the amount of a population ofprogenitor cells or their progeny needed to prevent or alleviate atleast one or more signs or symptoms of autosomal dominant CORD, andrelates to a sufficient amount of a composition to provide the desiredeffect, e.g., to treat a subject having autosomal dominant CORD. Theterm “therapeutically effective amount” therefore refers to an amount ofprogenitor cells or a composition comprising progenitor cells that issufficient to promote a particular effect when administered to a typicalsubject, such as one who has or is at risk for autosomal dominant CORD.An effective amount would also include an amount sufficient to preventor delay the development of a symptom of the disease, alter the courseof a symptom of the disease (for example but not limited to, slow theprogression of a symptom of the disease), or reverse a symptom of thedisease. It is understood that for any given case, an appropriate“effective amount” can be determined by one of ordinary skill in the artusing routine experimentation.

For use in the various aspects described herein, an effective amount ofprogenitor cells comprises at least 10² progenitor cells, at least 5×10²progenitor cells, at least 10³ progenitor cells, at least 5×10³progenitor cells, at least 10⁴ progenitor cells, at least 5×10⁴progenitor cells, at least 10⁵ progenitor cells, at least 2×10⁵progenitor cells, at least 3×10⁵ progenitor cells, at least 4×10⁵progenitor cells, at least 5×10⁵ progenitor cells, at least 6×10⁵progenitor cells, at least 7×10⁵ progenitor cells, at least 8×10⁵progenitor cells, at least 9×10⁵ progenitor cells, at least 1×10⁶progenitor cells, at least 2×10⁶ progenitor cells, at least 3×10⁶progenitor cells, at least 4×10⁶ progenitor cells, at least 5×10⁶progenitor cells, at least 6×10⁶ progenitor cells, at least 7×10⁶progenitor cells, at least 8×10⁶ progenitor cells, at least 9×10⁶progenitor cells, or multiples thereof. The progenitor cells can bederived from one or more donors, or can be obtained from an autologoussource. In some examples described herein, the progenitor cells can beexpanded in culture prior to administration to a subject in needthereof.

Modest and incremental increases in the levels of functional RetGC1protein expressed in cells of patients having autosomal dominant CORDcan be beneficial for ameliorating one or more symptoms of the disease,for increasing long-term survival, and/or for reducing side effectsassociated with other treatments. Upon administration of such cells tohuman patients, the presence of progenitors that are producing increasedlevels of functional RetGC1 protein is beneficial. In some cases,effective treatment of a subject gives rise to at least about 3%, 5% or7% functional RetGC1 protein relative to total RetGC1 in the treatedsubject. In some examples, functional RetGC1 will be at least about 10%of total RetGC1. In some examples, functional RetGC1 protein will be atleast about 20% to 30% of total RetGC1 protein. Similarly, theintroduction of even relatively limited subpopulations of cells havingsignificantly elevated levels of functional RetGC1 protein can bebeneficial in various patients because in some situations normalizedcells will have a selective advantage relative to diseased cells.However, even modest levels of progenitors with elevated levels offunctional RetGC1 protein can be beneficial for ameliorating one or moreaspects of autosomal dominant CORD in patients. In some examples, about10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%,about 80%, about 90% or more of the photoreceptor cells or retinalprogenitor cells in patients to whom such cells are administered areproducing increased levels of functional RetGC1 protein.

“Administered” refers to the delivery of a progenitor cell compositioninto a subject by a method or route that results in at least partiallocalization of the cell composition at a desired site. A cellcomposition can be administered by any appropriate route that results ineffective treatment in the subject, i.e. administration results indelivery to a desired location in the subject where at least a portionof the composition delivered, i.e. at least 1×10⁴ cells are delivered tothe desired site for a period of time.

In one aspect of the method, the pharmaceutical composition can beadministered via a route such as, but not limited to, enteral (into theintestine), gastroenteral, epidural (into the dura matter), oral (by wayof the mouth), transdermal, peridural, intracerebral (into thecerebrum), intracerebroventricular (into the cerebral ventricles),epicutaneous (application onto the skin), intradermal, (into the skinitself), subcutaneous (under the skin), nasal administration (throughthe nose), intravenous (into a vein), intravenous bolus, intravenousdrip, intraarterial (into an artery), intramuscular (into a muscle),intracardiac (into the heart), intraosseous infusion (into the bonemarrow), intrathecal (into the spinal canal), intraperitoneal, (infusionor injection into the peritoneum), intravesical infusion, intravitreal,(through the eye), intracavernous injection (into a pathologic cavity)intracavitary (into the base of the penis), intravaginal administration,intrauterine, extra-amniotic administration, transdermal (diffusionthrough the intact skin for systemic distribution), transmucosal(diffusion through a mucous membrane), transvaginal, insufflation(snorting), sublingual, sublabial, enema, eye drops (onto theconjunctiva), in ear drops, auricular (in or by way of the ear), buccal(directed toward the cheek), conjunctival, cutaneous, dental (to a toothor teeth), electro-osmosis, endocervical, endosinusial, endotracheal,extracorporeal, hemodialysis, infiltration, interstitial,intra-abdominal, intra-amniotic, intra-articular, intrabiliary,intrabronchial, intrabursal, intracartilaginous (within a cartilage),intracaudal (within the cauda equine), intracisternal (within thecisterna magna cerebellomedularis), intracorneal (within the cornea),dental intracornal, intracoronary (within the coronary arteries),intracorporus cavernosum (within the dilatable spaces of the corporuscavernosa of the penis), intradiscal (within a disc), intraductal(within a duct of a gland), intraduodenal (within the duodenum),intradural (within or beneath the dura), intraepidermal (to theepidermis), intraesophageal (to the esophagus), intragastric (within thestomach), intragingival (within the gingivae), intraileal (within thedistal portion of the small intestine), intralesional (within orintroduced directly to a localized lesion), intraluminal (within a lumenof a tube), intralymphatic (within the lymph), intramedullary (withinthe marrow cavity of a bone), intrameningeal (within the meninges),intramyocardial (within the myocardium), intraocular (within the eye),intraovarian (within the ovary), intrapericardial (within thepericardium), intrapleural (within the pleura), intraprostatic (withinthe prostate gland), intrapulmonary (within the lungs or its bronchi),intrasinal (within the nasal or periorbital sinuses), intraspinal(within the vertebral column), intrasynovial (within the synovial cavityof a joint), intratendinous (within a tendon), intratesticular (withinthe testicle), intrathecal (within the cerebrospinal fluid at any levelof the cerebrospinal axis), intrathoracic (within the thorax),intratubular (within the tubules of an organ), intratumor (within atumor), intratympanic (within the aurus media), intravascular (within avessel or vessels), intraventricular (within a ventricle), iontophoresis(by means of electric current where ions of soluble salts migrate intothe tissues of the body), irrigation (to bathe or flush open wounds orbody cavities), laryngeal (directly upon the larynx), nasogastric(through the nose and into the stomach), occlusive dressing technique(topical route administration, which is then covered by a dressing thatoccludes the area), ophthalmic (to the external eye), oropharyngeal(directly to the mouth and pharynx), parenteral, percutaneous,periarticular, peridural, perineural, periodontal, rectal, respiratory(within the respiratory tract by inhaling orally or nasally for local orsystemic effect), retrobulbar (behind the pons or behind the eyeball),intramyocardial (entering the myocardium), soft tissue, subarachnoid,subconjunctival, submucosal, topical, transplacental (through or acrossthe placenta), transtracheal (through the wall of the trachea),transtympanic (across or through the tympanic cavity), ureteral (to theureter), urethral (to the urethra), vaginal, caudal block, diagnostic,nerve block, biliary perfusion, cardiac perfusion, photopheresis andspinal.

Modes of administration include injection, infusion, instillation,and/or ingestion. “Injection” includes, without limitation, intravenous,intramuscular, intra-arterial, intrathecal, intraventricular,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular,subarachnoid, intraspinal, intracerebro spinal, and intrasternalinjection and infusion. In some examples, the route is intravenous. Forthe delivery of cells, administration by injection or infusion can bemade.

The cells can be administered systemically. The phrases “systemicadministration,” “administered systemically”, “peripheraladministration” and “administered peripherally” refer to theadministration of a population of progenitor cells other than directlyinto a target site, tissue, or organ, such that it enters, instead, thesubject's circulatory system and, thus, is subject to metabolism andother like processes.

The efficacy of a treatment comprising a composition for the treatmentof autosomal dominant CORD can be determined by the skilled clinician.However, a treatment is considered “effective treatment,” if any one orall of the signs or symptoms of, as but one example, levels offunctional autosomal dominant CORD are altered in a beneficial manner(e.g., increased by at least 10%), or other clinically accepted symptomsor markers of disease are improved or ameliorated. Efficacy can also bemeasured by failure of an individual to worsen as assessed byhospitalization or need for medical interventions (e.g., progression ofthe disease is halted or at least slowed). Methods of measuring theseindicators are known to those of skill in the art and/or describedherein. Treatment includes any treatment of a disease in an individualor an animal (some non-limiting examples include a human, or a mammal)and includes: (1) inhibiting the disease, e.g., arresting, or slowingthe progression of symptoms; or (2) relieving the disease, e.g., causingregression of symptoms; and (3) preventing or reducing the likelihood ofthe development of symptoms.

The treatment according to the present disclosure can ameliorate one ormore symptoms associated with autosomal dominant CORD by increasing,decreasing or altering the amount of functional RetGC1 in theindividual. Signs typically associated with autosomal dominant CORDinclude for example, decreased central vision, color vision defects,photophobia and decreased sensitivity in the central field at earlystages, followed by progressive loss in peripheral vision and nightblindness at later stages.

Kits

The present disclosure provides kits for carrying out the methodsdescribed herein. A kit can include one or more of a genome-targetingnucleic acid, a polynucleotide encoding a genome-targeting nucleic acid,a site-directed polypeptide, a polynucleotide encoding a site-directedpolypeptide, and/or any nucleic acid or proteinaceous molecule necessaryto carry out the aspects of the methods described herein, or anycombination thereof.

A kit can comprise: (1) a vector comprising a nucleotide sequenceencoding a genome-targeting nucleic acid, (2) the site-directedpolypeptide or a vector comprising a nucleotide sequence encoding thesite-directed polypeptide, and (3) a reagent for reconstitution and/ordilution of the vector(s) and or polypeptide.

A kit can comprise: (1) a vector comprising (i) a nucleotide sequenceencoding a genome-targeting nucleic acid, and (ii) a nucleotide sequenceencoding the site-directed polypeptide; and (2) a reagent forreconstitution and/or dilution of the vector.

In any of the above kits, the kit can comprise a single-molecule guidegenome-targeting nucleic acid. In any of the above kits, the kit cancomprise a double-molecule genome-targeting nucleic acid. In any of theabove kits, the kit can comprise two or more double-molecule guides orsingle-molecule guides. The kits can comprise a vector that encodes thenucleic acid targeting nucleic acid.

In any of the above kits, the kit can further comprise a polynucleotideto be inserted to effect the desired genetic modification.

Components of a kit can be in separate containers, or combined in asingle container.

Any kit described above can further comprise one or more additionalreagents, where such additional reagents are selected from a buffer, abuffer for introducing a polypeptide or polynucleotide into a cell, awash buffer, a control reagent, a control vector, a control RNApolynucleotide, a reagent for in vitro production of the polypeptidefrom DNA, adaptors for sequencing and the like. A buffer can be astabilization buffer, a reconstituting buffer, a diluting buffer, or thelike. A kit can also comprise one or more components that can be used tofacilitate or enhance the on-target binding or the cleavage of DNA bythe endonuclease, or improve the specificity of targeting.

In addition to the above-mentioned components, a kit can furthercomprise instructions for using the components of the kit to practicethe methods. The instructions for practicing the methods can be recordedon a suitable recording medium. For example, the instructions can beprinted on a substrate, such as paper or plastic, etc. The instructionscan be present in the kits as a package insert, in the labeling of thecontainer of the kit or components thereof (i.e., associated with thepackaging or sub packaging), etc. The instructions can be present as anelectronic storage data file present on a suitable computer readablestorage medium, e.g. CD-ROM, diskette, flash drive, etc. In someinstances, the actual instructions are not present in the kit, but meansfor obtaining the instructions from a remote source (e.g. via theInternet), can be provided. An example of this case is a kit thatcomprises a web address where the instructions can be viewed and/or fromwhich the instructions can be downloaded. As with the instructions, thismeans for obtaining the instructions can be recorded on a suitablesubstrate.

Additional Therapeutic Approaches

Gene editing can be conducted using nucleases engineered to targetspecific sequences. To date there are four major types of nucleases:meganucleases and their derivatives, zinc finger nucleases (ZFNs),transcription activator like effector nucleases (TALENs), andCRISPR-Cas9 nuclease systems. The nuclease platforms vary in difficultyof design, targeting density and mode of action, particularly as thespecificity of ZFNs and TALENs is through protein-DNA interactions,while RNA-DNA interactions primarily guide Cas9. Cas9 cleavage alsorequires an adjacent motif, the PAM, which differs between differentCRISPR systems. Cas9 from Streptococcus pyogenes cleaves using a NGGPAM, CRISPR from Neisseria meningitidis can cleave at sites with PAMsincluding NNNNGATT, NNNNNGTTT and NNNNGCTT. A number of other Cas9orthologs target protospacer adjacent to alternative PAMs.

CRISPR endonucleases, such as Cas9, can be used in the methods of thepresent disclosure. However, the teachings described herein, such astherapeutic target sites, could be applied to other forms ofendonucleases, such as ZFNs, TALENs, HEs, or MegaTALs, or usingcombinations of nulceases. However, in order to apply the teachings ofthe present disclosure to such endonucleases, one would need to, amongother things, engineer proteins directed to the specific target sites.

Additional binding domains can be fused to the Cas9 protein to increasespecificity. The target sites of these constructs would map to theidentified gRNA specified site, but would require additional bindingmotifs, such as for a zinc finger domain. In the case of Mega-TAL, ameganuclease can be fused to a TALE DNA-binding domain. The meganucleasedomain can increase specificity and provide the cleavage. Similarly,inactivated or dead Cas9 (dCas9) can be fused to a cleavage domain andrequire the sgRNA/Cas9 target site and adjacent binding site for thefused DNA-binding domain. This likely would require some proteinengineering of the dCas9, in addition to the catalytic inactivation, todecrease binding without the additional binding site.

Zinc Finger Nucleases

Zinc finger nucleases (ZFNs) are modular proteins comprised of anengineered zinc finger DNA binding domain linked to the catalytic domainof the type II endonuclease FokI. Because FokI functions only as adimer, a pair of ZFNs must be engineered to bind to cognate target“half-site” sequences on opposite DNA strands and with precise spacingbetween them to enable the catalytically active FokI dimer to form. Upondimerization of the FokI domain, which itself has no sequencespecificity per se, a DNA double-strand break is generated between theZFN half-sites as the initiating step in genome editing.

The DNA binding domain of each ZFN is typically comprised of 3-6 zincfingers of the abundant Cys2-His2 architecture, with each fingerprimarily recognizing a triplet of nucleotides on one strand of thetarget DNA sequence, although cross-strand interaction with a fourthnucleotide also can be important. Alteration of the amino acids of afinger in positions that make key contacts with the DNA alters thesequence specificity of a given finger. Thus, a four-finger zinc fingerprotein will selectively recognize a 12 bp target sequence, where thetarget sequence is a composite of the triplet preferences contributed byeach finger, although triplet preference can be influenced to varyingdegrees by neighboring fingers. An important aspect of ZFNs is that theycan be readily re-targeted to almost any genomic address simply bymodifying individual fingers, although considerable expertise isrequired to do this well. In most applications of ZFNs, proteins of 4-6fingers are used, recognizing 12-18 bp respectively. Hence, a pair ofZFNs will typically recognize a combined target sequence of 24-36 bp,not including the typical 5-7 bp spacer between half-sites. The bindingsites can be separated further with larger spacers, including 15-17 bp.A target sequence of this length is likely to be unique in the humangenome, assuming repetitive sequences or gene homologs are excludedduring the design process. Nevertheless, the ZFN protein-DNAinteractions are not absolute in their specificity so off-target bindingand cleavage events do occur, either as a heterodimer between the twoZFNs, or as a homodimer of one or the other of the ZFNs. The latterpossibility has been effectively eliminated by engineering thedimerization interface of the FokI domain to create “plus” and “minus”variants, also known as obligate heterodimer variants, which can onlydimerize with each other, and not with themselves. Forcing the obligateheterodimer prevents formation of the homodimer. This has greatlyenhanced specificity of ZFNs, as well as any other nuclease that adoptsthese FokI variants.

A variety of ZFN-based systems have been described in the art,modifications thereof are regularly reported, and numerous referencesdescribe rules and parameters that are used to guide the design of ZFNs;see, e.g., Segal et al., Proc Natl Acad Sci USA 96(6):2758-63 (1999);Dreier B et al., J Mol Biol. 303(4):489-502 (2000); Liu Q et al., J BiolChem. 277(6):3850-6 (2002); Dreier et al., J Biol Chem 280(42):35588-97(2005); and Dreier et al., J Biol Chem. 276(31):29466-78 (2001).

Transcription Activator-Like Effector Nucleases (TALENs)

Transcription Activator-Like Effector Nucleases (TALENs) representanother format of modular nucleases whereby, as with ZFNs, an engineeredDNA binding domain is linked to the FokI nuclease domain, and a pair ofTALENs operates in tandem to achieve targeted DNA cleavage. The majordifference from ZFNs is the nature of the DNA binding domain and theassociated target DNA sequence recognition properties. The TALEN DNAbinding domain derives from TALE proteins, which were originallydescribed in the plant bacterial pathogen Xanthomonas sp. TALEs arecomprised of tandem arrays of 33-35 amino acid repeats, with each repeatrecognizing a single base pair in the target DNA sequence that istypically up to 20 bp in length, giving a total target sequence lengthof up to 40 bp. Nucleotide specificity of each repeat is determined bythe repeat variable diresidue (RVD), which includes just two amino acidsat positions 12 and 13. The bases guanine, adenine, cytosine and thymineare predominantly recognized by the four RVDs: Asn-Asn, Asn-Ile, His-Aspand Asn-Gly, respectively. This constitutes a much simpler recognitioncode than for zinc fingers, and thus represents an advantage over thelatter for nuclease design. Nevertheless, as with ZFNs, the protein-DNAinteractions of TALENs are not absolute in their specificity, and TALENshave also benefitted from the use of obligate heterodimer variants ofthe FokI domain to reduce off-target activity.

Additional variants of the FokI domain have been created that aredeactivated in their catalytic function. If one half of either a TALENor a ZFN pair contains an inactive FokI domain, then only single-strandDNA cleavage (nicking) will occur at the target site, rather than a DSB.The outcome is comparable to the use of CRISPR/Cas9/Cpf1 “nickase”mutants in which one of the Cas9 cleavage domains has been deactivated.DNA nicks can be used to drive genome editing by HDR, but at lowerefficiency than with a DSB. The main benefit is that off-target nicksare quickly and accurately repaired, unlike the DSB, which is prone toNHEJ-mediated mis-repair.

A variety of TALEN-based systems have been described in the art, andmodifications thereof are regularly reported; see, e.g., Boch, Science326(5959):1509-12 (2009); Mak et al., Science 335(6069):716-9 (2012);and Moscou et al., Science 326(5959):1501 (2009). The use of TALENsbased on the “Golden Gate” platform, or cloning scheme, has beendescribed by multiple groups; see, e.g., Cermak et al., Nucleic AcidsRes. 39(12):e82 (2011); Li et al., Nucleic Acids Res.39(14):6315-25(2011); Weber et al., PLoS One. 6(2): e16765 (2011); Wanget al., J Genet Genomics 41(6):339-47, Epub 2014 May 17 (2014); andCermak T et al., Methods Mol Biol. 1239:133-59 (2015).

Homing Endonucleases

Homing endonucleases (HEs) are sequence-specific endonucleases that havelong recognition sequences (14-44 base pairs) and cleave DNA with highspecificity—often at sites unique in the genome. There are at least sixknown families of HEs as classified by their structure, includingLAGLIDADG (SEQ ID NO: 5271), GIY-YIG, His-Cis box, H—N—H, PD-(D/E)xK,and Vsr-like that are derived from a broad range of hosts, includingeukarya, protists, bacteria, archaea, cyanobacteria and phage. As withZFNs and TALENs, HEs can be used to create a DSB at a target locus asthe initial step in genome editing. In addition, some natural andengineered HEs cut only a single strand of DNA, thereby functioning assite-specific nickases. The large target sequence of HEs and thespecificity that they offer have made them attractive candidates tocreate site-specific DSBs.

A variety of HE-based systems have been described in the art, andmodifications thereof are regularly reported; see, e.g., the reviews bySteentoft et al., Glycobiology 24(8):663-80 (2014); Belfort andBonocora, Methods Mol Biol. 1123:1-26 (2014); Hafez and Hausner, Genome55(8):553-69 (2012).

MegaTAL/Tev-mTALEN/MegaTev

As further examples of hybrid nucleases, the MegaTAL platform andTev-mTALEN platform use a fusion of TALE DNA binding domains andcatalytically active HEs, taking advantage of both the tunable DNAbinding and specificity of the TALE, as well as the cleavage sequencespecificity of the HE; see, e.g., Boissel et al., NAR 42: 2591-2601(2014); Kleinstiver et al., G3 4:1155-65 (2014); and Boissel andScharenberg, Methods Mol. Biol. 1239: 171-96 (2015).

In a further variation, the MegaTev architecture is the fusion of ameganuclease (Mega) with the nuclease domain derived from the GIY-YIGhoming endonuclease I-TevI (Tev). The two active sites are positioned˜30 bp apart on a DNA substrate and generate two DSBs withnon-compatible cohesive ends; see, e.g., Wolfs et al., NAR 42, 8816-29(2014). It is anticipated that other combinations of existingnuclease-based approaches will evolve and be useful in achieving thetargeted genome modifications described herein.

dCas9-FokI or dCpf1-FokI and Other Nucleases

Combining the structural and functional properties of the nucleaseplatforms described above offers a further approach to genome editingthat can potentially overcome some of the inherent deficiencies. As anexample, the CRISPR genome editing system typically uses a single Cas9endonuclease to create a DSB. The specificity of targeting is driven bya 20 or 24 nucleotide sequence in the guide RNA that undergoesWatson-Crick base-pairing with the target DNA (plus an additional 2bases in the adjacent NAG or NGG PAM sequence in the case of Cas9 fromS. pyogenes). Such a sequence is long enough to be unique in the humangenome, however, the specificity of the RNA/DNA interaction is notabsolute, with significant promiscuity sometimes tolerated, particularlyin the 5′ half of the target sequence, effectively reducing the numberof bases that drive specificity. One solution to this has been tocompletely deactivate the Cas9 or Cpf1 catalytic function—retaining onlythe RNA-guided DNA binding function—and instead fusing a FokI domain tothe deactivated Cas9; see, e.g., Tsai et al., Nature Biotech 32: 569-76(2014); and Guilinger et al., Nature Biotech. 32: 577-82 (2014). BecauseFokI must dimerize to become catalytically active, two guide RNAs arerequired to tether two FokI fusions in close proximity to form the dimerand cleave DNA. This essentially doubles the number of bases in thecombined target sites, thereby increasing the stringency of targeting byCRISPR-based systems.

As further example, fusion of the TALE DNA binding domain to acatalytically active HE, such as I-TevI, takes advantage of both thetunable DNA binding and specificity of the TALE, as well as the cleavagesequence specificity of I-TevI, with the expectation that off-targetcleavage can be further reduced.

Methods and Compositions of the Invention

Accordingly, the present disclosure relates in particular to thefollowing non-limiting inventions:

In a first method, Method 1, the present disclosure provides a methodfor editing a GUCY2D gene in a human cell, the method comprising:introducing into the human cell one or more DNA endonucleases to effectone or more SSBs or DSBs within or near the GUCY2D gene or other DNAsequences that encode regulatory elements of the GUCY2D gene thatresults in a deletion, insertion, or correction thereby creating anedited human cell.

In another method, Method 2, the present disclosure provides a methodfor editing a R838H, R838C, or R838S mutation in a GUCY2D gene in ahuman cell, the method comprising: introducing into the human cell oneor more DNA endonucleases to effect one or more SSBs or DSBs within ornear the R838H, R838C, or R838S mutation in a GUCY2D gene that resultsin a deletion, insertion, or correction thereby creating an edited humancell.

In another method, Method 3, the present disclosure provides an in vivomethod for treating a patient with autosomal dominant CORD, the methodcomprising: editing a R838H, R838C, or R838S mutation in a GUCY2D genein a cell of the patient.

In another method, Method 4, the present disclosure provides an in vivomethod for treating a patient with autosomal dominant CORD as providedin Method 3, wherein the editing comprises: introducing into the cellone or more DNA endonucleases to effect one or more SSBs or DSBs withinor near the R838H, R838C, or R838S mutation in a GUCY2D gene thatresults in a deletion, insertion, or correction and results inrestoration of RetGC1 protein activity.

In another method, Method 5, the present disclosure provides an in vivomethod for treating a patient with autosomal dominant CORD as providedin Methods 1-2 or 4, wherein the one or more DNA endonucleases is aCas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also knownas Csn1 and Csx12), Cas100, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2,Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6,Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15,Csf1, Csf2, Csf3, Csf4, or Cpf1 endonuclease; a homolog thereof, arecombination of the naturally occurring molecule thereof,codon-optimized thereof, or modified versions thereof, and combinationsthereof.

In another method, Method 6, the present disclosure provides an in vivomethod for treating a patient with autosomal dominant CORD as providedin Method 5, wherein the method comprises introducing into the cell oneor more polynucleotides encoding the one or more DNA endonucleases.

In another method, Method 7, the present disclosure provides an in vivomethod for treating a patient with autosomal dominant CORD as providedin Method 5, wherein the method comprises introducing into the cell oneor more RNAs encoding the one or more DNA endonucleases.

In another method, Method 8, the present disclosure provides an in vivomethod for treating a patient with autosomal dominant CORD as providedin Methods 6 or 7, wherein the one or more polynucleotides or one ormore RNAs is one or more modified polynucleotides or one or moremodified RNAs.

In another method, Method 9, the present disclosure provides an in vivomethod for treating a patient with autosomal dominant CORD as providedin Method 5, wherein the DNA endonuclease is one or more proteins orpolypeptides.

In another method, Method 10, the present disclosure provides an in vivomethod for treating a patient with autosomal dominant CORD as providedin Methods 1-9, wherein the method further comprises: introducing intothe cell one or more gRNAs.

In another method, Method 11, the present disclosure provides an in vivomethod for treating a patient with autosomal dominant CORD as providedin Method 10, wherein the one or more gRNAs are sgRNAs.

In another method, Method 12, the present disclosure provides an in vivomethod for treating a patient with autosomal dominant CORD as providedin Methods 10-11, wherein the one or more gRNAs or one or more sgRNAs isone or more modified gRNAs or one or more modified sgRNAs.

In another method, Method 13, the present disclosure provides an in vivomethod for treating a patient with autosomal dominant CORD as providedin Methods 9-11, wherein the one or more DNA endonucleases ispre-complexed with one or more gRNAs or one or more sgRNAs.

In another method, Method 14, the present disclosure provides an in vivomethod for treating a patient with autosomal dominant CORD as providedin Methods 1-13, further comprising: introducing into the cell apolynucleotide donor template comprising at least a portion of thewild-type GUCY2D gene, or cDNA.

In another method, Method 15, the present disclosure provides an in vivomethod for treating a patient with autosomal dominant CORD as providedin Method 14, wherein the at least a portion of the wild-type GUCY2Dgene or cDNA is exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7,exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15,exon 16, exon 17, exon 18, exon 19, exon 20, intronic regions, fragmentsor combinations thereof, or the entire GUCY2D gene or cDNA.

In another method, Method 16, the present disclosure provides an in vivomethod for treating a patient with autosomal dominant CORD as providedin Methods 14-15, wherein the donor template is either a single ordouble stranded polynucleotide.

In another method, Method 17, the present disclosure provides an in vivomethod for treating a patient with autosomal dominant CORD as providedin Methods 14-15, wherein the donor template has homologous arms to the17p13.1 region.

In another method, Method 18, the present disclosure provides an in vivomethod for treating a patient with autosomal dominant CORD as providedin Methods 2 or 4, further comprising: introducing into the cell onegRNA and a polynucleotide donor template comprising at least a portionof the wild-type GUCY2D gene; wherein the one or more DNA endonucleasesis one or more Cas9 or Cpf1 endonucleases that effect one SSB or DSB ata locus located within or near the R838H, R838C, or R838S mutation in aGUCY2D gene that facilitates insertion of a new sequence from thepolynucleotide donor template into the chromosomal DNA at the locus thatresults in a insertion or correction of the R838H, R838C, or R838Smutation in a GUCY2D gene; and wherein the gRNA comprises a spacersequence that is complementary to a segment of the locus located withinor near the R838H, R838C, or R838S mutation in a GUCY2D gene.

In another method, Method 19, the present disclosure provides an in vivomethod for treating a patient with autosomal dominant CORD as providedin Methods 2 or 4, further comprising: introducing into the cell one ormore gRNAs and a polynucleotide donor template comprising at least aportion of the wild-type GUCY2D gene; wherein the one or more DNAendonucleases is one or more Cas9 or Cpf1 endonucleases that effect apair of SSBs or DSBs, the first at a 5′ locus and the second at a 3′locus, within or near the R838H, R838C, or R838S mutation in a GUCY2Dgene that facilitates insertion of a new sequence from thepolynucleotide donor template into the chromosomal DNA between the 5′locus and the 3′ locus that results in a insertion or correction of thechromosomal DNA between the 5′ locus and the 3′ locus within or near theR838H, R838C, or R838S mutation in a GUCY2D gene; and wherein the firstgRNA comprises a spacer sequence that is complementary to a segment ofthe 5′ locus and the second gRNA comprises a spacer sequence that iscomplementary to a segment of the 3′ locus.

In another method, Method 20, the present disclosure provides an in vivomethod for treating a patient with autosomal dominant CORD as providedin Methods 18-19, wherein the one or more gRNAs are one or more sgRNAs.

In another method, Method 21, the present disclosure provides an in vivomethod for treating a patient with autosomal dominant CORD as providedin Methods 18-20, wherein the one or more gRNAs or one or more sgRNAs isone or more modified gRNAs or one or more modified sgRNAs.

In another method, Method 22, the present disclosure provides an in vivomethod for treating a patient with autosomal dominant CORD as providedin Methods 18-21, wherein the one or more DNA endonucleases ispre-complexed with one or more gRNAs or one or more sgRNAs.

In another method, Method 23, the present disclosure provides an in vivomethod for treating a patient with autosomal dominant CORD as providedin Methods 18-22, wherein the at least a portion of the wild-type GUCY2Dgene or cDNA is exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7,exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15,exon 16, exon 17, exon 18, exon 19, exon 20, intronic regions, fragmentsor combinations thereof, or the entire GUCY2D gene or cDNA.

In another method, Method 24, the present disclosure provides an in vivomethod for treating a patient with autosomal dominant CORD as providedin Methods 18-23, wherein the donor template is either a single ordouble stranded polynucleotide.

In another method, Method 25, the present disclosure provides an in vivomethod for treating a patient with autosomal dominant CORD as providedin Methods 18-24, wherein the donor template has homologous arms to the17p13.1 region.

In another method, Method 26, the present disclosure provides an in vivomethod for treating a patient with autosomal dominant CORD as providedin Methods 18-25, wherein the SSB or DSB are in the first exon, secondexon, third exon, fourth exon, fifth exon, sixth exon, seventh exon,eighth exon, ninth exon, tenth exon, eleventh exon, twelfth exon,thirteenth exon, fourteenth exon, fifteenth exon, sixteenth exon,seventeenth exon, eighteenth exon, nineteenth exon, twentieth exon, orcombinations thereof of the GUCY2D gene.

In another method, Method 27, the present disclosure provides an in vivomethod for treating a patient with autosomal dominant CORD as providedin Methods 10-13 or 20-22, wherein the gRNA or sgRNA is directed to oneor more pathological variant: R838H, R838C, or R838S.

In another method, Method 28, the present disclosure provides an in vivomethod for treating a patient with autosomal dominant CORD as providedin Methods 1-2 or 4-27, wherein the insertion or correction is by HDR.

In another method, Method 29, the present disclosure provides an in vivomethod for treating a patient with autosomal dominant CORD as providedin Methods 18-19, wherein the donor template has homologous arms to apathological variant R838H, R838C, or R838S.

In another method, Method 30, the present disclosure provides an in vivomethod for treating a patient with autosomal dominant CORD as providedin Methods 2 or 4, further comprising: introducing into the cell twogRNAs and a polynucleotide donor template comprising at least a portionof the wild-type GUCY2D gene; wherein the one or more DNA endonucleasesis one or more Cas9 or Cpf1 endonucleases that effect a pair of DSBs,the first at a 5′ DSB locus and the second at a 3′ DSB locus, within ornear the R838H, R838C, or R838S mutation in a GUCY2D gene that causes adeletion of the chromosomal DNA between the 5′ DSB locus and the 3′ DSBlocus that results in a deletion of the chromosomal DNA between the 5′DSB locus and the 3′ DSB locus within or near the R838H, R838C, or R838Smutation in a GUCY2D gene; and wherein the first guide RNA comprises aspacer sequence that is complementary to a segment of the 5′ DSB locusand the second guide RNA comprises a spacer sequence that iscomplementary to a segment of the 3′ DSB locus.

In another method, Method 31, the present disclosure provides an in vivomethod for treating a patient with autosomal dominant CORD as providedin Method 30, wherein the two gRNAs are two sgRNAs.

In another method, Method 32, the present disclosure provides an in vivomethod for treating a patient with autosomal dominant CORD as providedin Methods 30-31, wherein the two gRNAs or two sgRNAs are two modifiedgRNAs or two modified sgRNAs.

In another method, Method 33, the present disclosure provides an in vivomethod for treating a patient with autosomal dominant CORD as providedin Methods 30-32, wherein the one or more DNA endonucleases ispre-complexed with two gRNAs or two sgRNAs.

In another method, Method 34, the present disclosure provides an in vivomethod for treating a patient with autosomal dominant CORD as providedin Methods 30-33, wherein both the 5′ DSB and 3′ DSB are in or neareither the first exon, first intron, second exon, second intron, thirdexon, third intron, fourth exon, fourth intron, fifth exon, fifthintron, sixth exon, sixth intron, seventh exon, seventh intron, eighthexon, eighth intron, ninth exon, ninth intron, tenth exon, tenth intron,eleventh exon, eleventh intron, twelfth exon, twelfth intron, thirteenthexon, thirteenth intron, fourteenth exon, fourteenth intron, fifteenthexon, fifteenth intron, sixteenth exon, sixteenth intron, seventeenthexon, seventeenth intron, eighteenth exon, eighteenth intron, nineteenthexon, nineteenth intron, twentieth exon, or combinations thereof, of theGUCY2D gene.

In another method, Method 35, the present disclosure provides an in vivomethod for treating a patient with autosomal dominant CORD as providedin Methods 30-34, wherein the deletion is a deletion of 1 kb or less.

In another method, Method 36, the present disclosure provides an in vivomethod for treating a patient with autosomal dominant CORD as providedin Methods 18-19 and 30, wherein the Cas9 or Cpf1 mRNA, gRNA, and donortemplate are either each formulated into separate lipid nanoparticles orall co-formulated into a lipid nanoparticle.

In another method, Method 37, the present disclosure provides an in vivomethod for treating a patient with autosomal dominant CORD as providedin Methods 18-19 and 30, wherein the Cas9 or Cpf1 mRNA, gRNA, and donortemplate are either each formulated into separate AAV vectors or allco-formulated into an AAV vector.

In another method, Method 38, the present disclosure provides an in vivomethod for treating a patient with autosomal dominant CORD as providedin Methods 18-19 and 30, wherein the Cas9 or Cpf1 mRNA is formulatedinto a lipid nanoparticle, and both the gRNA and donor template aredelivered to the cell by an AAV vector.

In another method, Method 39, the present disclosure provides an in vivomethod for treating a patient with autosomal dominant CORD as providedin Methods 18-19 and 30, wherein the Cas9 or Cpf1 mRNA is formulatedinto a lipid nanoparticle, and the gRNA is delivered to the cell byelectroporation and donor template is delivered to the cell by an AAVvector.

In another method, Method 40, the present disclosure provides an in vivomethod for treating a patient with autosomal dominant CORD as providedin Methods 37-39, wherein the AAV vector is a self-inactivating AAVvector.

In another method, Method 41, the present disclosure provides an in vivomethod for treating a patient with autosomal dominant CORD as providedin Methods 1-40, wherein the GUCY2D gene is located on Chromosome 17:8,002,594 to 8,020,339 (Genome Reference Consortium—GRCh38/hg38).

In another method, Method 42, the present disclosure provides an in vivomethod for treating a patient with autosomal dominant CORD as providedin Methods 2 or 4-41, wherein the restoration of RetGC1 protein activityis compared to wild-type or normal RetGC1 protein activity.

In another method, Method 43, the present disclosure provides a methodfor editing a GUCY2D gene in a human cell as provided in Methods 1-2,wherein the human cell is a photoreceptor cell or retinal progenitorcell.

In another method, Method 44, the present disclosure provides an in vivomethod for treating a patient with autosomal dominant CORD as providedin Methods 3-42, wherein the cell is a photoreceptor cell or retinalprogenitor cell.

In another method, Method 45, the present disclosure provides an in vivomethod for treating a patient with autosomal dominant CORD as providedin Method 14, wherein the polynucleotide donor template comprises exon 1of GUCY2D and is up to 5 KB.

In another method, Method 46, the present disclosure provides an in vivomethod for treating a patient with autosomal dominant CORD as providedin Method 45, wherein the polynucleotide donor template is delivered byAAV.

In another method, Method 47, the present disclosure provides a methodfor editing a R838H mutation in a GUCY2D gene in a human cell, themethod comprising: introducing into the human cell one or more DNAendonucleases to effect one or more SSBs or DSBs within or near theR838H mutation in a GUCY2D gene that results in a deletion, insertion,correction, or modulation of expression or function of the R838Hmutation thereby creating an edited human cell.

In another method, Method 48, the present disclosure provides a methodfor editing a R838C mutation in a GUCY2D gene in a human cell, themethod comprising: introducing into the human cell one or more DNAendonucleases to effect one or more SSBs or DSBs within or near theR838C mutation in a GUCY2D gene that results in a deletion, insertion,correction, or modulation of expression or function of the R838Cmutation thereby creating an edited human cell.

In another method, Method 49, the present disclosure provides a methodfor editing a R838S mutation in a GUCY2D gene in a human cell, themethod comprising: introducing into the human cell one or more DNAendonucleases to effect one or more SSBs or DSBs within or near theR838S mutation in a GUCY2D gene that results in a deletion, insertion,correction, or modulation of expression or function of the R838Smutation thereby creating an edited human cell.

In another method, Method 50, the present disclosure provides an in vivomethod for treating a patient with autosomal dominant CORD, the methodcomprising: editing a R838H mutation in a GUCY2D gene in a cell of thepatient.

In another method, Method 51, the present disclosure provides an in vivomethod for treating a patient with autosomal dominant CORD, the methodcomprising: editing a R838C mutation in a GUCY2D gene in a cell of thepatient.

In another method, Method 52, the present disclosure provides an in vivomethod for treating a patient with autosomal dominant CORD, the methodcomprising: editing a R838S mutation in a GUCY2D gene in a cell of thepatient.

In another method, Method 53, the present disclosure provides a methodfor editing an R838H mutation within a GUCY2D gene, the methodcomprising administering a gRNA or sgRNA comprising SEQ ID NO: 5285.

In another method, Method 54, the present disclosure provides a methodfor editing an R838H mutation within a GUCY2D gene, the methodcomprising administering a gRNA or sgRNA comprising SEQ ID NO: 5286.

In another method, Method 55, the present disclosure provides a methodfor editing an R838H mutation or R838C mutation within a GUCY2D gene,the method comprising administering a gRNA or sgRNA comprising SEQ IDNO: 5398.

In another method, Method 56, the present disclosure provides a methodfor editing an R838H mutation within a GUCY2D gene, the methodcomprising administering a gRNA or sgRNA comprising SEQ ID NO: 5464.

In another method, Method 57, the present disclosure provides a methodfor editing an R838H mutation within a GUCY2D gene, the methodcomprising administering a gRNA or sgRNA comprising SEQ ID NO: 5465.

In another method, Method 58, the present disclosure provides a methodfor editing an R838H mutation or R838C mutation within a GUCY2D gene,the method comprising administering a gRNA or sgRNA comprising SEQ IDNO: 5466.

In another method, Method 59, the present disclosure provides a methodfor treating a patient with an R838H mutation within a GUCY2D gene, themethod comprising administering a gRNA or sgRNA comprising SEQ ID NO:5285 to the patient.

In another method, Method 60, the present disclosure provides a methodfor treating a patient with an R838H mutation within a GUCY2D gene, themethod comprising administering a gRNA or sgRNA comprising SEQ ID NO:5286 to the patient.

In another method, Method 61, the present disclosure provides a methodfor treating a patient with an R838H mutation or R838C mutation within aGUCY2D gene, the method comprising administering a gRNA or sgRNAcomprising SEQ ID NO: 5398 to the patient.

In another method, Method 62, the present disclosure provides a methodfor treating a patient with an R838H mutation within a GUCY2D gene, themethod comprising administering a gRNA or sgRNA comprising SEQ ID NO:5464 to the patient.

In another method, Method 63, the present disclosure provides a methodfor treating a patient with an R838H mutation within a GUCY2D gene, themethod comprising administering a gRNA or sgRNA comprising SEQ ID NO:5465 to the patient.

In another method, Method 64, the present disclosure provides a methodfor treating a patient with an R838H mutation or R838C mutation within aGUCY2D gene, the method comprising administering a gRNA or sgRNAcomprising SEQ ID NO: 5466 to the patient.

In another method, Method 65, the present disclosure provides a methodfor editing an R838H mutation or R838C mutation within a GUCY2D gene,the method comprising administering the self-inactivating CRISPR-Cassystem of any of Self-inactivating CRISPR-Cas systems 1-35.

In another method, Method 66, the present disclosure provides a methodfor treating a patient with an R838H mutation or R838C mutation within aGUCY2D gene, the method comprising administering the self-inactivatingCRISPR-Cas system of any of Self-inactivating CRISPR-Cas systems 1-35.

In another method, Method 67, the present disclosure provides a methodof controlling Cas9 expression in a cell comprising: contacting the cellwith the the self-inactivating CRISPR-Cas system of any ofSelf-inactivating CRISPR-Cas systems 1-35.

In another method, Method 68, the present disclosure provides a methodfor editing a GUCY2D gene in a human cell as provided in Method 1,wherein the human cell has defective activity and the edited human cellexpresses a functional GUCY2D.

In another method, Method 69, the present disclosure provides a methodfor editing a R838H, R838C, or R838S mutation in a GUCY2D gene in ahuman cell as provided in Method 2, wherein the human cell has defectiveactivity and the edited human cell expresses a functional GUCY2D.

In another method, Method 70, the present disclosure provides a methodfor editing a R838H mutation in a GUCY2D gene in a human cell asprovided in Method 47, wherein the human cell has defective activity andthe edited human cell expresses a functional GUCY2D.

In another method, Method 71, the present disclosure provides a methodfor editing a R838C mutation in a GUCY2D gene in a human cell asprovided in Method 48, wherein the human cell has defective activity andthe edited human cell expresses a functional GUCY2D.

In another method, Method 72, the present disclosure provides a methodfor editing a R838S mutation in a GUCY2D gene in a human cell asprovided in Method 49, wherein the human cell has defective activity andthe edited human cell expresses a functional GUCY2D.

In another method, Method 73, the present disclosure provides a methodfor editing a GUCY2D gene in a human cell as provided in Method 1,wherein the deletion, insertion, or correction results in a modulationof expression or function of the GUCY2D gene.

In another method, Method 74, the present disclosure provides a methodfor editing a R838H, R838C, or R838S mutation in a GUCY2D gene in ahuman cell as provided in Method 2, wherein the deletion, insertion, orcorrection results in a modulation of expression or function of theGUCY2D gene.

In another method, Method 75, the present disclosure provides an in vivomethod for treating a patient with autosomal dominant CORD as providedin Method 4, wherein the deletion, insertion, or correction results in amodulation of expression or function of the GUCY2D gene and results inrestoration of retinal membrane guanylate cyclase-1 (RetGC1) proteinactivity.

In another method, Method 76, the present disclosure provides an in vivomethod for treating a patient with autosomal dominant CORD as providedin Method 3, wherein the editing comprises: introducing into the cellone or more DNA endonucleases to effect one or more SSBs or DSBs withinor near the R838H, R838C, or R838S mutation in a GUCY2D gene thatresults in a modulation of expression or function of the GUCY2D gene andresults in restoration of retinal membrane guanylate cyclase-1 (RetGC1)protein activity.

In another method, Method 77, the present disclosure provides a methodfor editing a GUCY2D gene in a human cell, the method comprising:introducing into the human cell one or more deoxyribonucleic acid (DNA)endonucleases to effect one or more single-strand breaks (SSBs) ordouble-strand breaks (DSBs) within or near the GUCY2D gene or other DNAsequences that encode regulatory elements of the GUCY2D gene thatresults in a modulation of expression or function of the GUCY2D genethereby creating an edited human cell.

In another method, Method 78, the present disclosure provides a methodfor editing a R838H, R838C, or R838S mutation in a GUCY2D gene in ahuman cell, the method comprising: introducing into the human cell oneor more DNA endonucleases to effect one or more SSBs or DSBs within ornear the R838H, R838C, or R838S mutation in a GUCY2D gene that resultsin a modulation of expression or function of the GUCY2D gene therebycreating an edited human cell.

In another method, Method 79, the present disclosure provides a methodfor editing a R838H mutation in a GUCY2D gene in a human cell, themethod comprising: introducing into the human cell one or more DNAendonucleases to effect one or more SSBs or DSBs within or near theR838H mutation in a GUCY2D gene that results in a deletion, insertion,or correction thereby creating an edited human cell.

In another method, Method 80, the present disclosure provides a methodfor editing a R838H mutation in a GUCY2D gene in a human cell, themethod comprising: introducing into the human cell one or more DNAendonucleases to effect one or more SSBs or DSBs within or near theR838H mutation in a GUCY2D gene that results in a modulation ofexpression or function thereby creating an edited human cell.

In another method, Method 81, the present disclosure provides a methodfor editing a R838C mutation in a GUCY2D gene in a human cell, themethod comprising: introducing into the human cell one or more DNAendonucleases to effect one or more SSBs or DSBs within or near theR838C mutation in a GUCY2D gene that results in a deletion, insertion,or correction thereby creating an edited human cell.

In another method, Method 82, the present disclosure provides a methodfor editing a R838C mutation in a GUCY2D gene in a human cell, themethod comprising: introducing into the human cell one or more DNAendonucleases to effect one or more SSBs or DSBs within or near theR838C mutation in a GUCY2D gene that results in a modulation ofexpression or function thereby creating an edited human cell.

In another method, Method 83, the present disclosure provides a methodfor editing a R838S mutation in a GUCY2D gene in a human cell, themethod comprising: introducing into the human cell one or more DNAendonucleases to effect one or more SSBs or DSBs within or near theR838S mutation in a GUCY2D gene that results in a deletion, insertion,or correction thereby creating an edited human cell.

In another method, Method 84, the present disclosure provides a methodfor editing a R838S mutation in a GUCY2D gene in a human cell, themethod comprising: introducing into the human cell one or more DNAendonucleases to effect one or more SSBs or DSBs within or near theR838S mutation in a GUCY2D gene that results in a modulation ofexpression or function thereby creating an edited human cell.

In a first composition, Composition 1, the present disclosure providesone or more gRNAs for editing a R838H, R838C, or R838S mutation in aGUCY2D gene in a cell from a patient with autosomal dominant CORD, theone or more gRNAs comprising a spacer sequence selected from the groupconsisting of nucleic acid sequences in SEQ ID NOs: 5282-5313,5398-5409, and 5434-5443 of the Sequence Listing.

In another composition, Composition 2, the present disclosure providesthe one or more gRNAs of Composition 1, wherein the one or more gRNAsare one or more sgRNAs.

In another composition, Composition 3, the present disclosure providesthe one or more gRNAs of Compositions 1 or 2, wherein the one or moregRNAs or one or more sgRNAs is one or more modified gRNAs or one or moremodified sgRNAs.

In another composition, Composition 4, the present disclosure providesthe one or more gRNAs of Compositions 1-3, wherein the cell is aphotoreceptor cell, retinal progenitor cell, or induced pluripotent stemcell (iPSC).

In another composition, Composition 5, the present disclosure providesone or more gRNAs for editing a R838H mutation in a GUCY2D gene in acell from a patient with autosomal dominant CORD, the one or more gRNAscomprising a spacer sequence selected from the group consisting ofnucleic acid sequences in SEQ ID NOs: 5282-5293, 5398-5409, and5434-5443 of the Sequence Listing.

In another composition, Composition 6, the present disclosure providesone or more gRNAs for editing a R838C mutation in a GUCY2D gene in acell from a patient with autosomal dominant CORD, the one or more gRNAscomprising a spacer sequence selected from the group consisting ofnucleic acid sequences in SEQ ID NOs: 5294-5303 and 5398-5409 of theSequence Listing.

In another composition, Composition 7, the present disclosure providesone or more gRNAs for editing a R838S mutation in a GUCY2D gene in acell from a patient with autosomal dominant CORD, the one or more gRNAscomprising a spacer sequence selected from the group consisting ofnucleic acid sequences in SEQ ID NOs: 5304-5313 and 5434-5443 of theSequence Listing.

In another composition, Composition 8, the present disclosure provides agRNA for editing a R838H or R838C mutation in a GUCY2D gene in a cellfrom a patient with autosomal dominant CORD, the gRNA comprising aspacer sequence selected from the group consisting of nucleic acidsequences in SEQ ID NOs: 5398-5409 of the Sequence Listing.

In another composition, Composition 9, the present disclosure providesthe gRNA of Composition 8, wherein the gRNA is a sgRNA.

In another composition, Composition 10, the present disclosure providesthe gRNA or sgRNA of Compositions 8 or 9, wherein the gRNA or sgRNA is amodified gRNA or modified sgRNA.

In another composition, Composition 11, the present disclosure providesthe gRNA or sgRNA of Compositions 8-10, wherein the cell is aphotoreceptor cell, retinal progenitor cell, or induced pluripotent stemcell (iPSC).

In another composition, Composition 12, the present disclosure providesa gRNA for editing a R838H or R838S mutation in a GUCY2D gene in a cellfrom a patient with autosomal dominant CORD, the gRNA comprising aspacer sequence selected from the group consisting of nucleic acidsequences in SEQ ID NOs: 5434-5443 of the Sequence Listing.

In another composition, Composition 13, the present disclosure providesthe gRNA of Composition 12, wherein the gRNA is a sgRNA.

In another composition, Composition 14, the present disclosure providesthe gRNA or sgRNA of Compositions 12 or 13, wherein the gRNA or sgRNA isa modified gRNA or modified sgRNA.

In another composition, Composition 15, the present disclosure providesthe gRNA or sgRNA of Compositions 12-14, wherein the cell is aphotoreceptor cell, retinal progenitor cell, or induced pluripotent stemcell (iPSC).

In another composition, Composition 16, the present disclosure providesa single-molecule guide RNA (sgRNA) for editing a R838H mutation in aGUCY2D gene in a cell from a patient with autosomal dominant CORD, thesgRNA comprising the nucleic acid sequence of SEQ ID NO: 5285.

In another composition, Composition 17, the present disclosure providesa single-molecule guide RNA (sgRNA) for editing a R838H or R838Cmutation in a GUCY2D gene in a cell from a patient with autosomaldominant CORD, the sgRNA comprising the nucleic acid sequence of SEQ IDNO: 5398.

In another composition, Composition 18, the present disclosure providesa single-molecule guide RNA (sgRNA) for editing a R838H mutation in aGUCY2D gene in a cell from a patient with autosomal dominant CORD, thesgRNA comprising the nucleic acid sequence of SEQ ID NO: 5286.

In another composition, Composition 19, the present disclosure providesa single-molecule guide RNA (sgRNA) for editing a R838H mutation in aGUCY2D gene in a cell from a patient with autosomal dominant CORD, thesgRNA comprising the nucleic acid sequence of SEQ ID NO: 5464.

In another composition, Composition 20, the present disclosure providesa single-molecule guide RNA (sgRNA) for editing a R838H mutation in aGUCY2D gene in a cell from a patient with autosomal dominant CORD, thesgRNA comprising the nucleic acid sequence of SEQ ID NO: 5465.

In another composition, Composition 21, the present disclosure providesa single-molecule guide RNA (sgRNA) for editing a R838H or R838Cmutation in a GUCY2D gene in a cell from a patient with autosomaldominant CORD, the sgRNA comprising the nucleic acid sequence of SEQ IDNO: 5466.

In another composition, Composition 22, the present disclosure providesone or more gRNAs for editing a R838H, R838C, or R838S mutation in aGUCY2D gene, the one or more gRNAs comprising a spacer sequence selectedfrom the group consisting of nucleic acid sequences in SEQ ID NOs:5282-5313, 5398-5409, and 5434-5443 of the Sequence Listing.

In a first therapeutic, Therapeutic 1, the present disclosure provides atherapeutic for treating a patient with autosomal dominant Cone-RodDystrophy, the therapeutic comprising at least one or more gRNAs forediting a R838H, R838C, or R838S mutation in a GUCY2D gene, the one ormore gRNAs comprising a spacer sequence selected from the groupconsisting of nucleic acid sequences in SEQ ID NOs: 5282-5313,5398-5409, and 5434-5443 of the Sequence Listing.

In another therapeutic, Therapeutic 2, the present disclosure providesthe therapeutic of Therapeutic 2, wherein the one or more gRNAs are oneor more sgRNAs.

In another therapeutic, Therapeutic 3, the present disclosure providesthe therapeutic of Therapeutics 1 or 2, wherein the one or more gRNAs orone or more sgRNAs is one or more modified gRNAs or one or more modifiedsgRNAs.

In another therapeutic, Therapeutic 4, the present disclosure provides atherapeutic for treating a patient with autosomal dominant CORD, thetherapeutic formed by a method comprising: introducing one or more DNAendonucleases; introducing one or more gRNA or one or more sgRNA forediting a R838H, R838C, or R838S mutation in a GUCY2D gene; andoptionally introducing one or more donor template; wherein the one ormore gRNAs or sgRNAs comprise a spacer sequence selected from the groupconsisting of nucleic acid sequences in SEQ ID NOs: 5282-5313,5398-5409, and 5434-5443 of the Sequence Listing.

In another therapeutic, Therapeutic 5, the present disclosure provides atherapeutic comprising at least one or more gRNAs for editing a R838Hmutation in a GUCY2D gene, the one or more gRNAs comprising a spacersequence selected from the group consisting of nucleic acid sequences inSEQ ID NOs: 5282-5293, 5398-5409, and 5434-5443 of the Sequence Listing.

In another therapeutic, Therapeutic 6, the present disclosure provides atherapeutic comprising at least one or more gRNAs for editing a R838Cmutation in a GUCY2D gene, the one or more gRNAs comprising a spacersequence selected from the group consisting of nucleic acid sequences inSEQ ID NOs: 5294-5303 and 5398-5409 of the Sequence Listing.

In another therapeutic, Therapeutic 7, the present disclosure provides atherapeutic comprising at least one or more gRNAs for editing a R838Smutation in a GUCY2D gene, the one or more gRNAs comprising a spacersequence selected from the group consisting of nucleic acid sequences inSEQ ID NOs: 5304-5313 and 5434-5443 of the Sequence Listing.

In another therapeutic, Therapeutic 8, the present disclosure provides atherapeutic for treating a patient with autosomal dominant CORD, formedby a method comprising: introducing one or more DNA endonucleases;introducing one or more gRNA or one or more sgRNA for editing a R838Hmutation in a GUCY2D gene; and optionally introducing one or more donortemplate; wherein the one or more gRNAs or sgRNAs comprise a spacersequence selected from the group consisting of nucleic acid sequences inSEQ ID NOs: 5282-5293, 5398-5409, and 5434-5443 of the Sequence Listing.

In another therapeutic, Therapeutic 9, the present disclosure provides atherapeutic for treating a patient with autosomal dominant CORD, formedby a method comprising: introducing one or more DNA endonucleases;introducing one or more gRNA or one or more sgRNA for editing a R838Cmutation in a GUCY2D gene; and optionally introducing one or more donortemplate; wherein the one or more gRNAs or sgRNAs comprise a spacersequence selected from the group consisting of nucleic acid sequences inSEQ ID NOs: 5294-5303 and 5398-5409 of the Sequence Listing.

In another therapeutic, Therapeutic 10, the present disclosure providesa therapeutic for treating a patient with autosomal dominant CORD,formed by a method comprising: introducing one or more DNAendonucleases; introducing one or more gRNA or one or more sgRNA forediting a R838S mutation in a GUCY2D gene; and optionally introducingone or more donor template; wherein the one or more gRNAs or sgRNAscomprise a spacer sequence selected from the group consisting of nucleicacid sequences in SEQ ID NOs: 5304-5313 and 5434-5443 of the SequenceListing.

In another therapeutic, Therapeutic 11, the present disclosure providesa therapeutic comprising a gRNA for editing a R838H or R838C mutation ina GUCY2D gene, the gRNA comprising a spacer sequence selected from thegroup consisting of nucleic acid sequences in SEQ ID NOs: 5398-5409 ofthe Sequence Listing.

In another therapeutic, Therapeutic 12, the present disclosure providesthe therapeutic of Therapeutic 11, wherein the gRNA is a sgRNA.

In another therapeutic, Therapeutic 13, the present disclosure providesthe therapeutic of Therapeutics 11 or 12, wherein the gRNA or sgRNA is amodified gRNA or modified sgRNA.

In another therapeutic, Therapeutic 14, the present disclosure providesa therapeutic for treating a patient with autosomal dominant CORD,formed by the method comprising: introducing one or more DNAendonucleases; introducing a gRNA or sgRNA for editing a R838H or R838Cmutation in a GUCY2D gene; and introducing one or more donor template;wherein the gRNA or sgRNA comprise a spacer sequence selected from thegroup consisting of nucleic acid sequences in SEQ ID NOs: 5398-5409 ofthe Sequence Listing.

In another therapeutic, Therapeutic 15, the present disclosure providesa therapeutic comprising a gRNA for editing a R838H or R838S mutation ina GUCY2D gene, the gRNA comprising a spacer sequence selected from thegroup consisting of nucleic acid sequences in SEQ ID NOs: 5434-5443 ofthe Sequence Listing.

In another therapeutic, Therapeutic 16, the present disclosure providesthe therapeutic of Therapeutic 15, wherein the gRNA is a sgRNA.

In another therapeutic, Therapeutic 17, the present disclosure providesthe therapeutic of Therapeutics 15 or 16, wherein the gRNA or sgRNA is amodified gRNA or modified sgRNA.

In another therapeutic, Therapeutic 18, the present disclosure providesa therapeutic for treating a patient with autosomal dominant CORD,formed by the method comprising: introducing one or more DNAendonucleases; introducing a gRNA or sgRNA for editing a R838H or R838Smutation in a GUCY2D gene; and introducing one or more donor template;wherein the gRNA or sgRNA comprise a spacer sequence selected from thegroup consisting of nucleic acid sequences in SEQ ID NOs: 5434-5443 ofthe Sequence Listing.

In another therapeutic, Therapeutic 19, the present disclosure providesa therapeutic comprising the self-inactivating CRISPR-Cas system of anyof Self-inactivating CRISPR-Cas systems 1-35.

In another therapeutic, Therapeutic 20, the present disclosure providesthe therapeutic of Therapeutic 19, wherein the therapeutic is sterile.

In a first kit, Kit 1, the present disclosure provides a kit fortreating a patient with autosomal dominant CORD in vivo, the kitcomprising one or more gRNAs or sgRNAs for editing a R838H, R838C, orR838S mutation in a GUCY2D gene wherein the one or more gRNAs or sgRNAscomprise a spacer sequence selected from the group consisting of nucleicacid sequences in SEQ ID NOs: 5282-5313, 5398-5409, and 5434-5443 of theSequence Listing; one or more DNA endonucleases; and optionally, one ormore donor template.

In another kit, Kit 2, the present disclosure provides the kit of Kit 1,wherein the one or more DNA endonucleases is a Cas1, Cas1B, Cas2, Cas3,Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12),Cas100, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2,Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3,Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3,Csf4, or Cpf1 endonuclease; a homolog thereof, a recombination of thenaturally occurring molecule thereof, codon-optimized thereof, ormodified versions thereof, and combinations thereof.

In another kit, Kit 3, the present disclosure provides the kit of Kits 1or 2, comprising one or more donor template.

In another kit, Kit 4, the present disclosure provides the kit of Kit 3,wherein the donor template has homologous arms to the 17p13.1 region.

In another kit, Kit 5, the present disclosure provides the kit of Kit 3,wherein the donor template has homologous arms to a pathological variantR838H, R838C, or R838S.

In another kit, Kit 6, the present disclosure provides a kit fortreating a patient with autosomal dominant CORD in vivo, the kitcomprising one or more gRNAs or sgRNAs for editing a R838H mutation in aGUCY2D gene wherein the one or more gRNAs or sgRNAs comprise a spacersequence selected from the group consisting of nucleic acid sequences inSEQ ID NOs: 5282-5293, 5398-5409, and 5434-5443 of the Sequence Listing;one or more DNA endonucleases; and optionally, one or more donortemplate.

In another kit, Kit 7, the present disclosure provides a kit fortreating a patient with autosomal dominant CORD in vivo, the kitcomprising one or more gRNAs or sgRNAs for editing a R838C mutation in aGUCY2D gene wherein the one or more gRNAs or sgRNAs comprise a spacersequence selected from the group consisting of nucleic acid sequences inSEQ ID NOs: 5294-5303 and 5398-5409 of the Sequence Listing; one or moreDNA endonucleases; and optionally, one or more donor template.

In another kit, Kit 8, the present disclosure provides a kit fortreating a patient with autosomal dominant CORD in vivo, the kitcomprising one or more gRNAs or sgRNAs for editing a R838S mutation in aGUCY2D gene wherein the one or more gRNAs or sgRNAs comprise a spacersequence selected from the group consisting of nucleic acid sequences inSEQ ID NOs: 5304-5313 and 5434-5443 of the Sequence Listing; one or moreDNA endonucleases; and optionally, one or more donor template.

In another kit, Kit 9, the present disclosure provides a kit fortreating a patient with autosomal dominant CORD in vivo, the kitcomprising a gRNA or sgRNA for editing a R838H or R838C mutation in aGUCY2D gene, wherein the gRNA or sgRNA comprise a spacer sequenceselected from the group consisting of nucleic acid sequences in SEQ IDNOs: 5398-5409 of the Sequence Listing; one or more DNA endonucleases;and optionally, one or more donor template.

In another kit, Kit 10, the present disclosure provides the kit of Kit9, wherein the one or more DNA endonucleases is a Cas1, Cas1B, Cas2,Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12),Cas100, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2,Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3,Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3,Csf4, or Cpf1 endonuclease; a homolog thereof, a recombination of thenaturally occurring molecule thereof, codon-optimized thereof, ormodified versions thereof, and combinations thereof.

In another kit, Kit 11, the present disclosure provides the kit of Kits9 or 10, comprising one or more donor template.

In another kit, Kit 12, the present disclosure provides the kit of Kit11, wherein the donor template has homologous arms to the 17p13.1region.

In another kit, Kit 13, the present disclosure provides the kit of Kit11, wherein the donor template has homologous arms to a pathologicalvariant R838H or R838C.

In another kit, Kit 14, the present disclosure provides a kit fortreating a patient with autosomal dominant CORD in vivo, the kitcomprising a gRNA or sgRNA for editing a R838H or R838S mutation in aGUCY2D gene, wherein the gRNA or sgRNA comprise a spacer sequenceselected from the group consisting of nucleic acid sequences in SEQ IDNOs: 5434-5443 of the Sequence Listing; one or more DNA endonucleases;and optionally, one or more donor template.

In another kit, Kit 15, the present disclosure provides the kit of Kit14, wherein the one or more DNA endonucleases is a Cas1, Cas1B, Cas2,Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12),Cas100, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2,Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3,Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3,Csf4, or Cpf1 endonuclease; a homolog thereof, a recombination of thenaturally occurring molecule thereof, codon-optimized thereof, ormodified versions thereof, and combinations thereof.

In another kit, Kit 16, the present disclosure provides the kit of Kits14 or 15, comprising one or more donor template.

In another kit, Kit 17, the present disclosure provides the kit of Kit16, wherein the donor template has homologous arms to the 17p13.1region.

In another kit, Kit 18, the present disclosure provides the kit of Kit16, wherein the donor template has homologous arms to a pathologicalvariant R838H or R838S.

In another kit, Kit 19, the present disclosure provides a kit fortreating a patient with autosomal dominant CORD in vivo, the kitcomprising: any one of Self-inactivating CRISPR-Cas systems 1-35; andoptionally, one or more donor template.

In another kit, Kit 20, the present disclosure provides the kit of Kit19, comprising one or more donor template.

In another kit, Kit 21, the present disclosure provides the kit of Kit20, wherein the donor template has homologous arms to the 17p13.1region.

In another kit, Kit 22, the present disclosure provides the kit of Kit20, wherein the donor template has homologous arms to a pathologicalvariant R838H, R838C, or R838S.

In a first self-inactivating CRISPR-Cas system, Self-inactivatingCRISPR-Cas system 1, the present disclosure provides a self-inactivatingCRISPR-Cas system comprising: a first segment comprising a nucleotidesequence that encodes a polypeptide inducing site-directed mutagenesis;a second segment comprising a nucleotide sequence that encodes a guideRNA (gRNA) or a single-molecule guide RNA (sgRNA) wherein the gRNA orsgRNA comprise SEQ ID NO: 5285 or 5464; and one or more third segmentscomprising a self-inactivating (SIN) site; wherein the gRNA or sgRNA issubstantially complementary to the SIN site; wherein the gRNA or sgRNAis substantially complementary to a genomic target sequence.

In another self-inactivating CRISPR-Cas system, Self-inactivatingCRISPR-Cas system 2, the present disclosure provides a self-inactivatingCRISPR-Cas system comprising a first segment comprising a nucleotidesequence that encodes a a polypeptide inducing site-directedmutagenesis; a second segment comprising a nucleotide sequence thatencodes a guide RNA (gRNA) or single-molecule guide RNA (sgRNA) whereinthe gRNA or sgRNA comprise SEQ ID NO: 5398 or 5466; and one or morethird segments comprising a self-inactivating (SIN) site; wherein thegRNA or sgRNA is substantially complementary to the SIN site; whereinthe gRNA or sgRNA is substantially complementary to a genomic targetsequence.

In another self-inactivating CRISPR-Cas system, Self-inactivatingCRISPR-Cas system 3, the present disclosure provides theself-inactivating CRISPR-Cas system of Self-inactivating CRISPR-Cassystems 1 or 2, wherein the polypeptide inducing site-directedmutagenesis is Streptococcus pyogenes Cas9 (SpCas9) or any variantsthereof.

In another self-inactivating CRISPR-Cas system, Self-inactivatingCRISPR-Cas system 4, the present disclosure provides theself-inactivating CRISPR-Cas system of Self-inactivating CRISPR-Cassystems 1-3, wherein the polypeptide inducing site-directed mutagenesisis SpCas9 or any variants thereof; and wherein the SIN site is a 5′ SINsite located 5′ of a SpCas9 open reading frame (ORF) or a 3′ SIN sitelocated 3′ of the SpCas9 ORF.

In another self-inactivating CRISPR-Cas system, Self-inactivatingCRISPR-Cas system 5, the present disclosure provides theself-inactivating CRISPR-Cas system of Self-inactivating CRISPR-Cassystem 4, wherein the 5′ SIN site comprises SEQ ID NO: 5327.

In another self-inactivating CRISPR-Cas system, Self-inactivatingCRISPR-Cas system 6, the present disclosure provides theself-inactivating CRISPR-Cas system of Self-inactivating CRISPR-Cassystems 4-5, wherein the 3′ SIN site comprises SEQ ID NO: 5369.

In another self-inactivating CRISPR-Cas system, Self-inactivatingCRISPR-Cas system 7, the present disclosure provides theself-inactivating CRISPR-Cas system of Self-inactivating CRISPR-Cassystem 4, wherein the 5′ SIN site comprises SEQ ID NO: 5326.

In another self-inactivating CRISPR-Cas system, Self-inactivatingCRISPR-Cas system 8, the present disclosure provides theself-inactivating CRISPR-Cas system of Self-inactivating CRISPR-Cassystems 4 and 7, wherein the 3′ SIN site comprises SEQ ID NO: 5368.

In another self-inactivating CRISPR-Cas system, Self-inactivatingCRISPR-Cas system 9, the present disclosure provides theself-inactivating CRISPR-Cas system of Self-inactivating CRISPR-Cassystems 4-5 and 7, wherein the 5′ SIN site is located upstream of theCas9 open reading frame (ORF) and downstream of a SV40 nuclearlocalization signal (NLS).

In another self-inactivating CRISPR-Cas system, Self-inactivatingCRISPR-Cas system 10, the present disclosure provides theself-inactivating CRISPR-Cas system of Self-inactivating CRISPR-Cassystems 4-5 and 7, wherein the 5′ SIN site is located upstream of theCas9 open reading frame (ORF) and upstream of a SV40 nuclearlocalization signal (NLS) within a 5′ untranslated region (UTR).

In another self-inactivating CRISPR-Cas system, Self-inactivatingCRISPR-Cas system 11, the present disclosure provides theself-inactivating CRISPR-Cas system of Self-inactivating CRISPR-Cassystems 1-10, where the SIN site comprises a protospacer adjacent motif(PAM).

In another self-inactivating CRISPR-Cas system, Self-inactivatingCRISPR-Cas system 12, the present disclosure provides theself-inactivating CRISPR-Cas system of Self-inactivating CRISPR-Cassystem 11, wherein the PAM is NRG or any variants thereof.

In another self-inactivating CRISPR-Cas system, Self-inactivatingCRISPR-Cas system 13, the present disclosure provides theself-inactivating CRISPR-Cas system of Self-inactivating CRISPR-Cassystems 1-12, wherein the genomic target sequence is a R838H mutation orR838C mutation in a guanylate cyclase 2D (GUCY2D) gene.

In another self-inactivating CRISPR-Cas system, Self-inactivatingCRISPR-Cas system 14, the present disclosure provides theself-inactivating CRISPR-Cas system of Self-inactivating CRISPR-Cassystems 1-13, wherein the first segment comprising a nucleotide sequencethat encodes a polypeptide inducing site-directed mutagenesis, furthercomprises a start codon, a stop codon, and a poly(A) termination site.

In another self-inactivating CRISPR-Cas system, Self-inactivatingCRISPR-Cas system 15, the present disclosure provides theself-inactivating CRISPR-Cas system of Self-inactivating CRISPR-Cassystems 1-14, wherein the first segment and the third segment areprovided together in a first vector and the second segment is providedin a second vector.

In another self-inactivating CRISPR-Cas system, Self-inactivatingCRISPR-Cas system 16, the present disclosure provides theself-inactivating CRISPR-Cas system of Self-inactivating CRISPR-Cassystems 1-14, wherein the first segment, second segment, and thirdsegment are provided together in a vector.

In another self-inactivating CRISPR-Cas system, Self-inactivatingCRISPR-Cas system 17, the present disclosure provides theself-inactivating CRISPR-Cas system of Self-inactivating CRISPR-Cassystems 15-16, wherein the third segment is present in the first orsecond vector at a location 5′ of the first segment.

In another self-inactivating CRISPR-Cas system, Self-inactivatingCRISPR-Cas system 18, the present disclosure provides theself-inactivating CRISPR-Cas system of Self-inactivating CRISPR-Cassystems 15-16, wherein the third segment is present in the first orsecond vector at a location 3′ of the first segment.

In another self-inactivating CRISPR-Cas system, Self-inactivatingCRISPR-Cas system 19, the present disclosure provides theself-inactivating CRISPR-Cas system of Self-inactivating CRISPR-Cassystems 15-16, wherein the one or more third segments are present in thefirst or second vector at the 5′ and 3′ ends of the first segment.

In another self-inactivating CRISPR-Cas system, Self-inactivatingCRISPR-Cas system 20, the present disclosure provides theself-inactivating CRISPR-Cas system of Self-inactivating CRISPR-Cassystem 15, wherein the first vector comprises SEQ ID NO: 5508 and thesecond vector comprises SEQ ID NO: 5506.

In another self-inactivating CRISPR-Cas system, Self-inactivatingCRISPR-Cas system 21, the present disclosure provides theself-inactivating CRISPR-Cas system of Self-inactivating CRISPR-Cassystem 15, wherein the first vector comprises SEQ ID NO: 5508 and thesecond vector comprises SEQ ID NO: 5507.

In another self-inactivating CRISPR-Cas system, Self-inactivatingCRISPR-Cas system 22, the present disclosure provides theself-inactivating CRISPR-Cas system of Self-inactivating CRISPR-Cassystem 15, wherein the first vector comprises SEQ ID NO: 5509 and thesecond vector comprises SEQ ID NO: 5506.

In another self-inactivating CRISPR-Cas system, Self-inactivatingCRISPR-Cas system 23, the present disclosure provides theself-inactivating CRISPR-Cas system of Self-inactivating CRISPR-Cassystem 15, wherein the first vector comprises SEQ ID NO: 5509 and thesecond vector comprises SEQ ID NO: 5507.

In another self-inactivating CRISPR-Cas system, Self-inactivatingCRISPR-Cas system 24, the present disclosure provides theself-inactivating CRISPR-Cas system of Self-inactivating CRISPR-Cassystems 1-23, wherein the third segment is less than 100 nucleotides inlength.

In another self-inactivating CRISPR-Cas system, Self-inactivatingCRISPR-Cas system 25, the present disclosure provides theself-inactivating CRISPR-Cas system of Self-inactivating CRISPR-Cassystem 24, wherein the third segment is less than 50 nucleotides inlength.

In another self-inactivating CRISPR-Cas system, Self-inactivatingCRISPR-Cas system 26, the present disclosure provides theself-inactivating CRISPR-Cas system of Self-inactivating CRISPR-Cassystems 1-25, wherein the gRNA or sgRNA is fully complementary to thenucleotide sequence of the SIN site except for in at least one location.

In another self-inactivating CRISPR-Cas system, Self-inactivatingCRISPR-Cas system 27, the present disclosure provides theself-inactivating CRISPR-Cas system of Self-inactivating CRISPR-Cassystems 1-26, wherein the gRNA or sgRNA is fully complementary to thenucleotide sequence of the SIN site except for in at least twolocations.

In another self-inactivating CRISPR-Cas system, Self-inactivatingCRISPR-Cas system 28, the present disclosure provides theself-inactivating CRISPR-Cas system of Self-inactivating CRISPR-Cassystems 1-27, wherein a nucleic acid sequence encoding a promoter isoperably linked to the first segment.

In another self-inactivating CRISPR-Cas system, Self-inactivatingCRISPR-Cas system 29, the present disclosure provides theself-inactivating CRISPR-Cas system of Self-inactivating CRISPR-Cassystem 28, wherein the promoter is a spatially-restricted promoter,bidirectional promoter driving gRNA or sgRNA in one direction and SpCas9in the opposite orientation, or an inducible promoter.

In another self-inactivating CRISPR-Cas system, Self-inactivatingCRISPR-Cas system 30, the present disclosure provides theself-inactivating CRISPR-Cas system of Self-inactivating CRISPR-Cassystem 29, wherein the spatially-restricted promoter is selected fromthe group consisting of: any tissue or cell type specific promoter, ahepatocyte-specific promoter, a neuron-specific promoter, anadipocyte-specific promoter, a cardiomyocyte-specific promoter, askeletal muscle-specific promoter, lung progenitor cell specificpromoter, a photoreceptor-specific promoter, and a retinal pigmentepithelial (RPE) selective promoter.

In another self-inactivating CRISPR-Cas system, Self-inactivatingCRISPR-Cas system 31, the present disclosure provides theself-inactivating CRISPR-Cas system of Self-inactivating CRISPR-Cassystems 15-16, wherein the vector is one or more adeno-associated virus(AAV) vectors.

In another self-inactivating CRISPR-Cas system, Self-inactivatingCRISPR-Cas system 32, the present disclosure provides theself-inactivating CRISPR-Cas system of Self-inactivating CRISPR-Cassystem 31, wherein the adeno-associated virus (AAV) vector is an AAV5serotype capsid vector.

In another self-inactivating CRISPR-Cas system, Self-inactivatingCRISPR-Cas system 33, the present disclosure provides aself-inactivating CRISPR-Cas system comprising: a first segmentcomprising a nucleotide sequence that encodes a SpCas9 or any variantsthereof; a second segment comprising a nucleotide sequence that encodesa guide RNA (gRNA) or a single-molecule guide RNA (sgRNA); and one ormore third segments comprising a self-inactivating (SIN) site; whereinthe gRNA or sgRNA is substantially complementary to the SIN site;wherein the gRNA or sgRNA is substantially complementary to a genomictarget sequence; wherein the SIN site comprises a sequence selected fromthe group consisting of SEQ ID NOs: 5478-5492.

In another self-inactivating CRISPR-Cas system, Self-inactivatingCRISPR-Cas system 34, the present disclosure provides aself-inactivating CRISPR-Cas system comprising: a first segmentcomprising a nucleotide sequence that encodes a SpCas9 or any variantsthereof; a second segment comprising a nucleotide sequence that encodesa guide RNA (gRNA) or a single-molecule guide RNA (sgRNA); and one ormore third segments comprising a self-inactivating (SIN) site; whereinthe gRNA or sgRNA is substantially complementary to the SIN site;wherein the gRNA or sgRNA is substantially complementary to a genomictarget sequence; wherein the SIN site comprises a sequence selected fromthe group consisting of SEQ ID NOs: 5324-5355, 5410-5421 and 5444-5453or SEQ ID NOs: 5366-5397, 5422-5433, and 5454-5463.

In another self-inactivating CRISPR-Cas system, Self-InactivatingCRISPR-Cas system 35, the present disclosure provides aself-inactivating CRISPR-Cas system of Self-inactivating CRISPR-Cassystem 34, wherein the SIN site comprises a sequence 1, 2, or 3nucleotides shorter than any one of the sequences selected from thegroup consisting of SEQ ID NOs: 5324-5355, 5410-5421 and 5444-5453 orSEQ ID NOs: 5366-5397, 5422-5433, and 5454-5463.

In a first genetically modified cell, Genetically Modified Cell 1, thepresent disclosure provides a genetically modified cell comprising theself-inactivating CRISPR-Cas system of any of Self-inactivatingCRISPR-Cas systems 1-35.

In another genetically modified cell, Genetically Modified Cell 2, thepresent disclosure provides the genetically modified cell of GeneticallyModified Cell 1, wherein the cell is selected from the group consistingof: an archaeal cell, a bacterial cell, a eukaryotic cell, a eukaryoticsingle-cell organism, a somatic cell, a germ cell, a stem cell, a plantcell, an algal cell, an animal cell, an invertebrate cell, a vertebratecell, a fish cell, a frog cell, a bird cell, a mammalian cell, a pigcell, a cow cell, a goat cell, a sheep cell, a rodent cell, a rat cell,a mouse cell, a non-human primate cell, and a human cell.

In a first nucleic acid, Nucleic Acid 1, the present disclosure providesa nucleic acid encoding a gRNA comprising a spacer sequence selectedfrom the group consisting of SEQ ID NOs: 5282-5313, 5398-5409, and5434-5443.

In another nucleic acid, Nucleic Acid 2, the present disclosure providesthe nucleic acid of Nucleic Acid 1, wherein the gRNA is a sgRNA.

In a first vector, Vector 1, the present disclosure provides a vectorencoding a gRNA comprising a spacer sequence selected from the groupconsisting of SEQ ID NOs: 5282-5313, 5398-5409, and 5434-5443.

In another vector, Vector 2, the present disclosure provides the vectorof Vector 1, wherein the gRNA is a sgRNA.

In another vector, Vector 3, the present disclosure provides the vectorof any one of Vectors 1 or 2, wherein the vector is an AAV.

In another vector, Vector 4, the present disclosure provides the vectorof any one of Vectors 1-3, wherein the vector is an AAV5 sertoype capsidvector.

Definitions

In addition to the definitions previously set forth herein, thefollowing definitions are relevant to the present disclosure:

The term “alteration” or “alteration of genetic information” refers toany change in the genome of a cell. In the context of treating geneticdisorders, alterations may include, but are not limited to, insertion,deletion and correction.

The term “insertion” refers to an addition of one or more nucleotides ina DNA sequence. Insertions can range from small insertions of a fewnucleotides to insertions of large segments such as a cDNA or a gene.

The term “deletion” refers to a loss or removal of one or morenucleotides in a DNA sequence or a loss or removal of the function of agene. In some cases, a deletion can include, for example, a loss of afew nucleotides, an exon, an intron, a gene segment, or the entiresequence of a gene. In some cases, deletion of a gene refers to theelimination or reduction of the function or expression of a gene or itsgene product. This can result from not only a deletion of sequenceswithin or near the gene, but also other events (e.g., insertion,nonsense mutation) that disrupt the expression of the gene.

The term “correction” as used herein, refers to a change of one or morenucleotides of a genome in a cell, whether by insertion, deletion orsubstitution. Such correction may result in a more favorable genotypicor phenotypic outcome, whether in structure or function, to the genomicsite which was corrected. One non-limiting example of a “correction”includes the correction of a mutant or defective sequence to a wild-typesequence which restores structure or function to a gene or its geneproduct(s). Depending on the nature of the mutation, correction may beachieved via various strategies disclosed herein. In one non-limitingexample, a missense mutation may be corrected by replacing the regioncontaining the mutation with its wild-type counterpart. As anotherexample, duplication mutations (e.g., repeat expansions) in a gene maybe corrected by removing the extra sequences.

The term “knock-in” refers to an addition of a DNA sequence, or fragmentthereof into a genome. Such DNA sequences to be knocked-in may includean entire gene or genes, may include regulatory sequences associatedwith a gene or any portion or fragment of the foregoing. For example, acDNA encoding the wild-type protein may be inserted into the genome of acell carrying a mutant gene. Knock-in strategies need not replace thedefective gene, in whole or in part. In some cases, a knock-in strategymay further involve substitution of an existing sequence with theprovided sequence, e.g., substitution of a mutant allele with awild-type copy. On the other hand, the term “knock-out” refers to theelimination of a gene or the expression of a gene. For example, a genecan be knocked out by either a deletion or an addition of a nucleotidesequence that leads to a disruption of the reading frame. As anotherexample, a gene may be knocked out by replacing a part of the gene withan irrelevant sequence. Finally, the term “knock-down” as used hereinrefers to reduction in the expression of a gene or its gene product(s).As a result of a gene knock-down, the protein activity or function maybe attenuated or the protein levels may be reduced or eliminated.

The term “comprising” or “comprises” is used in reference tocompositions, therapeutics, kits, methods, and respective component(s)thereof, that are essential to the present disclosure, yet open to theinclusion of unspecified elements, whether essential or not.

The term “consisting essentially of” refers to those elements requiredfor a given aspect. The term permits the presence of additional elementsthat do not materially affect the basic and novel or functionalcharacteristic(s) of that aspect of the present disclosure.

The term “consisting of” refers to compositions, therapeutics, kits,methods, and respective components thereof as described herein, whichare exclusive of any element not recited in that description of theaspect.

The singular forms “a,” “an,” and “the” include plural references,unless the context clearly dictates otherwise.

Any numerical range recited in this specification describes allsub-ranges of the same numerical precision (i.e., having the same numberof specified digits) subsumed within the recited range. For example, arecited range of “1.0 to 10.0” describes all sub-ranges between (andincluding) the recited minimum value of 1.0 and the recited maximumvalue of 10.0, such as, for example, “2.4 to 7.6,” even if the range of“2.4 to 7.6” is not expressly recited in the text of the specification.Accordingly, the Applicant reserves the right to amend thisspecification, including the claims, to expressly recite any sub-rangeof the same numerical precision subsumed within the ranges expresslyrecited in this specification. All such ranges are inherently describedin this specification such that amending to expressly recite any suchsub-ranges will comply with written description, sufficiency ofdescription, and added matter requirements, including the requirementsunder 35 U.S.C. § 112(a) and Article 123(2) EPC. Also, unless expresslyspecified or otherwise required by context, all numerical parametersdescribed in this specification (such as those expressing values,ranges, amounts, percentages, and the like) may be read as if prefacedby the word “about,” even if the word “about” does not expressly appearbefore a number. Additionally, numerical parameters described in thisspecification should be construed in light of the number of reportedsignificant digits, numerical precision, and by applying ordinaryrounding techniques. It is also understood that numerical parametersdescribed in this specification will necessarily possess the inherentvariability characteristic of the underlying measurement techniques usedto determine the numerical value of the parameter.

Any patent, publication, or other disclosure material identified hereinis incorporated by reference into this specification in its entiretyunless otherwise indicated, but only to the extent that the incorporatedmaterial does not conflict with existing descriptions, definitions,statements, or other disclosure material expressly set forth in thisspecification. As such, and to the extent necessary, the expressdisclosure as set forth in this specification supersedes any conflictingmaterial incorporated by reference. Any material, or portion thereof,that is said to be incorporated by reference into this specification,but which conflicts with existing definitions, statements, or otherdisclosure material set forth herein, is only incorporated to the extentthat no conflict arises between that incorporated material and theexisting disclosure material. Applicants reserve the right to amend thisspecification to expressly recite any subject matter, or portionthereof, incorporated by reference herein.

The details of one or more aspects of the present disclosure are setforth in the accompanying examples below. Although any materials andmethods similar or equivalent to those described herein can be used inthe practice or testing of the present disclosure, specific examples ofthe materials and methods contemplated are now described. Otherfeatures, objects and advantages of the present disclosure will beapparent from the description. In the description examples, the singularforms also include the plural unless the context clearly dictatesotherwise. Unless defined otherwise, all technical and scientific termsused herein have the same meaning as commonly understood by one ofordinary skill in the art to which this present disclosure belongs. Inthe case of conflict, the present description will control.

EXAMPLES

The present disclosure will be more fully understood by reference to thefollowing examples, which provide illustrative non-limiting aspects ofthe invention.

The examples describe the use of the CRISPR system as an illustrativegenome editing technique to create defined therapeutic genomicdeletions, insertions, or replacements, termed “genomic modifications”herein, within or near the R838H, R838C, or R838S mutation in the GUCY2Dgene that lead to a frameshift and silencing of the expression of themutant gene or correction of the R838H, R838C, or R838S mutation in thegenomic locus, or expression at a heterologous locus, that restoreRetGC1 protein activity. Introduction of the defined therapeuticmodifications represents a novel therapeutic strategy for the potentialamelioration of autosomal dominant CORD, as described and illustratedherein.

Example 1—CRISPR/S. pyogenes (Sp) Cas9 PAM Sites for the R838H Mutationin the GUCY2D Gene

To discover target sites for genome editing by SpCas9, the R838Hmutation in the GUCY2D gene was scanned for SpCas9 protospacer adjacentmotifs (PAMs). The area was scanned for PAMs having the sequence NRG.gRNA spacer sequences (17-24 bps) located immediately upstream of theNRG PAM were then identified. These sequences are candidates for use inediting the gene.

Example 2—CRISPR/S. aureus (Sa) Cas9 PAM Sites for the R838H Mutation inthe GUCY2D Gene

To discover target sites for genome editing by SaCas9, the R838Hmutation in the GUCY2D gene was scanned for SaCas9 PAMs. The area wasscanned for PAMs having the sequence NNGRRT. gRNA spacer sequences(17-24 bps) located immediately upstream of the NNGRRT PAM were thenidentified. These sequences are candidates for use in editing the gene.

Example 3—CRISPR/S. thermophilus(St) Cas9 PAM Sites for the R838HMutation in the GUCY2D Gene

To discover target sites for genome editing by StCas9, the R838Hmutation in the GUCY2D gene is scanned for StCas9 PAMs. The area isscanned for PAMs having the sequence NNAGAAW. gRNA spacer sequences(17-24 bps) located immediately upstream of the NNAGAAW PAM are thenidentified. These sequences are candidates for use in editing the gene.

Example 4—CRISPR/T. denticola (Td) Cas9 PAM Sites for the R838H Mutationin the GUCY2D Gene

To discover target sites for genome editing by TdCas9, the R838Hmutation in the GUCY2D gene is scanned for TdCas9 PAMs. The area isscanned for PAMs having the sequence NAAAAC. gRNA spacer sequences(17-24 bps) located immediately upstream of the NAAAAC PAM are thenidentified. These sequences are candidates for use in editing the gene.

Example 5—CRISPR/N. meningitides (Nm) Cas9 PAM Sites for the R838HMutation in the GUCY2D Gene

To discover target sites for genome editing by NmCas9, the R838Hmutation in the GUCY2D gene is scanned for NmCas9 PAMs. The area isscanned for PAMs having the sequence NNNNGHTT. gRNA spacer sequences(17-24 bps) located immediately upstream of the NNNNGHTT PAM are thenidentified. These sequences are candidates for use in editing the gene.

Example 6—CRISPR/Cpf1 PAM Sites for the R838H Mutation in the GUCY2DGene

To discover target sites for genome editing by Cpf1, the R838H mutationin the GUCY2D gene is scanned for Cpf1 PAMs. The area is scanned forPAMs having the sequence YTN. gRNA spacer sequences (17-24 bps) locatedimmediately upstream of the YTN PAM are then identified. These sequencesare candidates for use in editing the gene.

Example 7—CRISPR/S. pyogenes (Sp) Cas9 PAM Sites for the R838C Mutationin the GUCY2D Gene

To discover target sites for genome editing by SpCas9, the R838Cmutation in the GUCY2D gene was scanned for SpCas9 PAMs. The area wasscanned for PAMs having the sequence NRG. gRNA spacer sequences (17-24bps) located immediately upstream of the NRG PAM were then identified.These sequences are candidates for use in editing the gene.

Example 8—CRISPR/S. aureus (Sa) Cas9 PAM Sites for the R838C Mutation inthe GUCY2D Gene

To discover target sites for genome editing by SaCas9, the R838Cmutation in the GUCY2D gene was scanned for SaCas9 PAMs. The area wasscanned for PAMs having the sequence NNGRRT. gRNA spacer sequences(17-24 bps) located immediately upstream of the NNGRRT PAM were thenidentified. These sequences are candidates for use in editing the gene.

Example 9—CRISPR/S. thermophilus(St) Cas9 PAM Sites for the R838CMutation in the GUCY2D Gene

To discover target sites for genome editing by StCas9, the R838Cmutation in the GUCY2D gene is scanned for StCas9 PAMs. The area isscanned for PAMs having the sequence NNAGAAW. gRNA spacer sequences(17-24 bps) located immediately upstream of the NNAGAAW PAM are thenidentified. These sequences are candidates for use in editing the gene.

Example 10—CRISPR/T. denticola (Td) Cas9 PAM Sites for the R838CMutation in the GUCY2D Gene

To discover target sites for genome editing by StCas9, the R838Cmutation in the GUCY2D gene is scanned for TdCas9 PAMs. The area isscanned for PAMs having the sequence NAAAAC. gRNA spacer sequences(17-24 bps) located immediately upstream of the NAAAAC PAM are thenidentified. These sequences are candidates for use in editing the gene.

Example 11—CRISPR/N. meningitides (Nm) Cas9 PAM Sites for the R838CMutation in the GUCY2D Gene

To discover target sites for genome editing by NmCas9, the R838Cmutation in the GUCY2D gene is scanned for NmCas9 PAMs. The area isscanned for PAMs having the sequence NNNNGHTT. gRNA spacer sequences(17-24 bps) located immediately upstream of the NNNNGHTT PAM are thenidentified. These sequences are candidates for use in editing the gene.

Example 12—CRISPR/Cpf1 PAM Sites for the R838C Mutation in the GUCY2DGene

To discover target sites for genome editing by Cpf1, the R838C mutationin the GUCY2D gene is scanned for Cpf1 PAMs. The area is scanned forPAMs having the sequence YTN. gRNA spacer sequences (17-24 bps) locatedimmediately upstream of the YTN PAM are then identified. These sequencesare candidates for use in editing the gene.

Example 13—CRISPR/S. pyogenes (Sp) Cas9 PAM Sites for the R838S Mutationin the GUCY2D Gene

To discover target sites for genome editing by SpCas9, the R838Smutation in the GUCY2D gene was scanned for SpCas9 PAMs. The area wasscanned for PAMs having the sequence NRG. gRNA spacer sequences (17-24bps) located immediately upstream of the NRG PAM were then identified.These sequences are candidates for use in editing the gene.

Example 14—CRISPR/S. aureus (Sa) Cas9 PAM Sites for the R838S Mutationin the GUCY2D Gene

To discover target sites for genome editing by SaCas9, the R838Smutation in the GUCY2D gene was scanned for SaCas9 PAMs. The area wasscanned for PAMs having the sequence NNGRRT. gRNA spacer sequences(17-24 bps) located immediately upstream of the NNGRRT PAM were thenidentified. These sequences are candidates for use in editing the gene.

Example 15—CRISPR/S. thermophilus (St) Cas9 PAM Sites for the R838SMutation in the GUCY2D Gene

To discover target sites for genome editing by StCas9, the R838Smutation in the GUCY2D gene is scanned for StCas9 PAMs. The area isscanned for PAMs having the sequence NNAGAAW. gRNA spacer sequences(17-24 bps) located immediately upstream of the NNAGAAW PAM are thenidentified. These sequences are candidates for use in editing the gene.

Example 16—CRISPR/T. denticola (Td) Cas9 PAM Sites for the R838SMutation in the GUCY2D Gene

To discover target sites for genome editing by TdCas9, the R838Smutation in the GUCY2D gene is scanned for TdCas9 PAMs. The area isscanned for PAMs having the sequence NAAAAC. gRNA spacer sequences(17-24 bps) located immediately upstream of the NAAAAC PAM are thenidentified. These sequences are candidates for use in editing the gene.

Example 17—CRISPR/N. meningitides (Nm) Cas9 PAM Sites for the R838SMutation in the GUCY2D Gene

To discover target sites for genome editing by NmCas9, the R838Smutation in the GUCY2D gene is scanned for NmCas9 PAMs. The area isscanned for PAMs having the sequence NNNNGHTT. gRNA spacer sequences(17-24 bps) located immediately upstream of the NNNNGHTT PAM are thenidentified. These sequences are candidates for use in editing the gene.

Example 18—CRISPR/Cpf1 PAM Sites for the R838S Mutation in the GUCY2DGene

To discover target sites for genome editing by Cpf1, the R838S mutationin the GUCY2D gene is scanned for Cpf1 PAMs. The area is scanned forPAMs having the sequence YTN. gRNA spacer sequences (17-24 bps) locatedimmediately upstream of the YTN PAM are then identified. These sequencesare candidates for use in editing the gene.

Example 19—Design of R838CH Double Mutation sgRNAs

One problem that can arise with gene editing is specificity. Aparticular challenge addressed by some examples provided herein is toinduce effective levels of editing in a targeted mutant allele whilemaintaining the integrity of wild type alleles. Furthermore, applicantswere able to design gRNAs that can be used to target multiple specificmutations.

To assess alternative gRNAs and decrease targeting of the wild-typeGUCY2D allele by gRNAs of the present disclosure, gRNAs that can directediting of either the R838C allele or the R838H allele were designed. Asdescribed in previous examples, PAMs and corresponding sgRNA spacersequences were identified for the R838C and R838H GUCY2D mutations.These two mutations occur in sequential base pairs in the GUCY2D gene,but typically do not both occur in a single patient. Applicants designedgRNAs or sgRNAs that can hybridize to (e.g., bind to) and target eithermutation with a single mismatch. For example, these “R838CH doublemutation” sgRNAs can hybridize with an R838C GUCY2D allele in a cell ofa first patient, or, separately, with an R838H GUCY2D allele in a cellof a second patient. In each case, there is a single mismatch betweenthe sgRNA and the mutant allele, but editing at the target locus stilloccurs.

An additional advantageous feature of such double mutation gRNAs orsgRNAs is that when hybridizing with a wild-type GUCY2D sequence, thedouble mutation gRNA or sgRNA, for example, an R838CH double mutationsgRNA, has two consecutive mismatches. The presence of two consecutivemismatches causes reduced off-target editing at the wild-type locus,compared to wild-type off-target editing caused by a single mutationsequence such as either R838C or R838H sgRNAs that bind to a wild-typeGUCY2D allele with only one mismatch.

Table 7 below shows various groupings of sgRNA spacer sequences that are19 or 20 nucleotides in length and that result in zero, one, or twomismatches when binding to the wild-type GUCY2D gene and zero or onemismatches when binding to a mutant GUCY2D allele. Bolded bases withinthe sgRNA sequence show the potential mismatch locations (which are alsothe individual bases altered by the various R838 mutations). The sgRNAspacer sequences in Table 7 are named for the allele to which they bindwith zero mismatches, except for the R838CH double mutation sgRNA spacersequences, which are named to show that they can bind either the R838Cor the R838H mutant allele with one mismatch.

TABLE 7 Single Guide SEQ RNA (sgRNA) ID GUCY2D Allele(s)Sequence (5′→3′) NOs WT GUCY2D 20mer UCUGAUCCCGGAGCGCACGG 5274WT GUCY2D 19mer CUGAUCCCGGAGCGCACGG 5278 R838H GUCY2D 20merUCUGAUCCCGGAGCACACGG 5284 R838H GUCY2D 19mer CUGAUCCCGGAGCACACGG 5289R838C GUCY2D 20mer UCUGAUCCCGGAGUGCACGG 5296 R838C GUCY2D 19merCUGAUCCCGGAGUGCACGG 5301 R838CH GUCY2D 20mer UCUGAUCCCGGAGUACACGG 5398R838CH GUCY2D 19mer CUGAUCCCGGAGUACACGG 5403 WT GUCY2D 20merGGAUCUGAUCCCGGAGCGCA 5275 WT GUCY2D 19mer GAUCUGAUCCCGGAGCGCA 5279R838H GUCY2D 20mer GGAUCUGAUCCCGGAGCACA 5285 R838H GUCY2D 19merGAUCUGAUCCCGGAGCACA 5290 R838C GUCY2D 20mer GGAUCUGAUCCCGGAGUGCA 5297R838C GUCY2D 19mer GAUCUGAUCCCGGAGUGCA 5300 R838CH GUCY2D 20merGGAUCUGAUCCCGGAGUACA 5399 R838CH GUCY2D 19mer GAUCUGAUCCCGGAGUACA 5404WT GUCY2D 20mer CCAGCUCCUCCGUGCGCUCC 5276 WT GUCY2D 19merCAGCUCCUCCGUGCGCUCC 5280 R838H GUCY2D 20mer CCAGCUCCUCCGUGUGCUCC 5286R838H GUCY2D 19mer CAGCUCCUCCGUGUGCUCC 5291 R838C GUCY2D 20merCCAGCUCCUCCGUGCACUCC 5298 R838C GUCY2D 19mer CAGCUCCUCCGUGCACUCC 5302R838CH GUCY2D 20mer CCAGCUCCUCCGUGUACUCC 5400 R838CH GUCY2D 19merCAGCUCCUCCGUGUACUCC 5405 R838H GUCY2D 20mer GCACACGGAGGAGCUGGAGC 5288R838H GUCY2D 19mer CACACGGAGGAGCUGGAGC 5293 R838CH GUCY2D 20merGUACACGGAGGAGCUGGAGC 5401 R838CH GUCY2D 19mer UACACGGAGGAGCUGGAGC 5406WT GUCY2D 20mer CCCGGAGCGCACGGAGGAGC 5277 WT GUCY2D 19merCCGGAGCGCACGGAGGAGC 5281 R838H GUCY2D 20mer CCCGGAGCACACGGAGGAGC 5287R838H GUCY2D 19mer CCGGAGCACACGGAGGAGC 5292 R838C GUCY2D 20merCCCGGAGUGCACGGAGGAGC 5299 R838C GUCY2D 19mer CCGGAGUGCACGGAGGAGC 5303R838CH GUCY2D 20mer CCCGGAGUACACGGAGGAGC 5402 R838CH GUCY2D 19merCCGGAGUACACGGAGGAGC 5407 WT GUCY2D 20mer UCCAGCUCCUCCGUGCGCUC 5272WT GUCY2D 19mer CCAGCUCCUCCGUGCGCUC 5273 R838H GUCY2D 20merUCCAGCUCCUCCGUGUGCUC 5282 R838H GUCY2D 19mer CCAGCUCCUCCGUGUGCUC 5283R838C GUCY2D 20mer UCCAGCUCCUCCGUGCACUC 5294 R838C GUCY2D 19merCCAGCUCCUCCGUGCACUC 5295 R838CH GUCY2D 20mer UCCAGCUCCUCCGUGUACUC 5408R838CH GUCY2D 19mer CCAGCUCCUCCGUGUACUC 5409

These R838CH double mutation sgRNAs were designed starting from spacersequences designed to target the R838H mutation (see Examples 1-2) andseparate spacer sequences designed to target the R838C mutation (seeExamples 7-8). For example, in the first grouping of Table 7, SEQ IDNOs: 5284 and 5296 are shown. SEQ ID NO: 5284 targets the R838H mutationand SEQ ID NO: 5296 targets the R838C mutation. A R838CH double mutationspacer sequence, such as SEQ ID NO: 5398, can be designed bymanipulating the bases shown in bold typeface in SEQ ID NOs: 5284 and5296. For example, SEQ ID NO: 5398 was designed by replacing the boldedC residue in SEQ ID NO: 5284 with the bolded U residue in SEQ ID NO:5296 to yield the bolded UA sequence in SEQ ID NO: 5398. SEQ ID NO: 5398could also be designed by starting with SEQ ID NO: 5296 and replacingthe bolded G residue with an A residue of SEQ ID NO: 5284 to yield thebolded UA sequence in SEQ ID NO: 5398. SEQ ID NOs: 5284, 5296, and 5398are all 20mer spacer sequences.

A similar process was used to generate the 19mer R838CH double mutationspacer sequence identified as SEQ ID NO: 5403, starting from SEQ ID NOs:5289 and 5301.

A similar process was used to generate the sgRNAs in the remaininggroupings in Table 7.

SEQ ID NOs: 5274 and 5278 (which bind to the wild-type GUCY2D allelewith zero mismatches) are also shown in the first grouping of Table 7 asa reference to the wild-type GUCY2D sequence.

SEQ ID NOs: 5398-5407 refer to sgRNA spacer sequences of R838CH doublemutation sgRNAs that associate with SpCas9. SEQ ID NOs: 5408-5409 referto sgRNA spacer sequences of R838CH double mutation sgRNAs thatassociate with SaCas9.

The R838CH double mutation gRNAs of the present disclosure can allow formore specific editing of the R838C and/or R838H mutant alleles whilereducing off-target editing of a wild type allele.

Example 20—Design of R838SH Double Mutation sgRNAs

To assess gRNAs and decrease targeting of the wild-type GUCY2D allele bygRNAs of the present disclosure, gRNAs that can direct editing of eitherthe R838S allele or the R838H allele were designed. As described inprevious examples, PAMs and corresponding sgRNA spacer sequences wereidentified for the R838S and R838H GUCY2D mutations. These two mutationsoccur in sequential base pairs in the GUCY2D gene, but typically do notboth occur in a single patient. Applicants designed gRNAs or sgRNAs thatcan hybridize to (e.g., bind to) and target either mutation with asingle mismatch. For example, these “R838SH” double mutation sgRNAs canhybridize with an R838S GUCY2D allele in a cell of a first patient, or,separately, with an R838H GUCY2D allele in a cell of a second patient.In each case, there is a single mismatch between the sgRNA and themutant allele, but editing at the target locus still occurs.

An additional advantageous feature of such double mutation gRNAs orsgRNAs is that when hybridizing with a wild-type GUCY2D sequence, thedouble mutation gRNA or sgRNA, for example, a R838SH double mutationsgRNA, has two consecutive mismatches. The presence of two consecutivemismatches causes reduced off-target editing at the wild-type locus,compared to wild-type off-target editing caused by a single mutationsequence such as either R838S or R838H sgRNAs that bind to a wild-typeGUCY2D allele with only one mismatch.

Table 8 below shows various groupings of sgRNA spacer sequences that are19 or 20 nucleotides in length and that result in zero, one, or twomismatches when binding to the wild-type GUCY2D gene and zero or onemismatches when binding to a mutant GUCY2D allele. Bolded bases withinthe sgRNA sequence show the potential mismatch locations (which are alsothe individual bases altered by the various R838 mutations). The sgRNAspacer sequences in Table 8 are named for the allele to which they bindwith zero mismatches, except for the R838SH double mutation sgRNA spacersequences, which are named to show that they can bind either the R838Sor the R838H mutant allele with one mismatch.

TABLE 8 Single Guide SEQ RNA (sgRNA) ID GUCY2D Allele(s)Sequence (5′→3′) NOs WT GUCY2D 20mer UCUGAUCCCGGAGCGCACGG 5274WT GUCY2D 19mer CUGAUCCCGGAGCGCACGG 5278 R838H GUCY2D 20merUCUGAUCCCGGAGCACACGG 5284 R838H GUCY2D 19mer CUGAUCCCGGAGCACACGG 5289R838S GUCY2D 20mer UCUGAUCCCGGAGAGCACGG 5304 R838S GUCY2D 19merCUGAUCCCGGAGAGCACGG 5309 R838SH GUCY2D 20mer UCUGAUCCCGGAGAACACGG 5437R838SH GUCY2D 19mer CUGAUCCCGGAGAACACGG 5440 WT GUCY2D 20merGGAUCUGAUCCCGGAGCGCA 5275 WT GUCY2D 19mer GAUCUGAUCCCGGAGCGCA 5279R838H GUCY2D 20mer GGAUCUGAUCCCGGAGCACA 5285 R838H GUCY2D 19merGAUCUGAUCCCGGAGCACA 5290 R838S GUCY2D 20mer GGAUCUGAUCCCGGAGAGCA 5305R838S GUCY2D 19mer GAUCUGAUCCCGGAGAGCA 5308 R838SH GUCY2D 20merGGAUCUGAUCCCGGAGAACA 5436 R838SH GUCY2D 19mer GAUCUGAUCCCGGAGAACA 5441WT GUCY2D 20mer CCAGCUCCUCCGUGCGCUCC 5276 WT GUCY2D 19merCAGCUCCUCCGUGCGCUCC 5280 R838H GUCY2D 20mer CCAGCUCCUCCGUGUGCUCC 5286R838H GUCY2D 19mer CAGCUCCUCCGUGUGCUCC 5291 R838S GUCY2D 20merCCAGCUCCUCCGUGCUCUCC 5306 R838S GUCY2D 19mer CAGCUCCUCCGUGCUCUCC 5310R838SH GUCY2D 20mer CCAGCUCCUCCGUGUUCUCC 5438 R838SH GUCY2D 19merCAGCUCCUCCGUGUUCUCC 5442 WT GUCY2D 20mer CCCGGAGCGCACGGAGGAGC 5277WT GUCY2D 19mer CCGGAGCGCACGGAGGAGC 5281 R838H GUCY2D 20merCCCGGAGCACACGGAGGAGC 5287 R838H GUCY2D 19mer CCGGAGCACACGGAGGAGC 5292R838S GUCY2D 20mer CCCGGAGAGCACGGAGGAGC 5307 R838S GUCY2D 19merCCGGAGAGCACGGAGGAGC 5311 R838SH GUCY2D 20mer CCCGGAGAACACGGAGGAGC 5439R838SH GUCY2D 19mer CCGGAGAACACGGAGGAGC 5443 WT GUCY2D 20merUCCAGCUCCUCCGUGCGCUC 5272 WT GUCY2D 19mer CCAGCUCCUCCGUGCGCUC 5273R838H GUCY2D 20mer UCCAGCUCCUCCGUGUGCUC 5282 R838H GUCY2D 19merCCAGCUCCUCCGUGUGCUC 5283 R838S GUCY2D 20mer UCCAGCUCCUCCGUGCUCUC 5312R838S GUCY2D 19mer CCAGCUCCUCCGUGCUCUC 5313 R838SH GUCY2D 20merUCCAGCUCCUCCGUGUUCUC 5434 R838SH GUCY2D 19mer CCAGCUCCUCCGUGUUCUC 5435

These R838SH double mutation sgRNAs were designed starting from spacersequences designed to target the R838H mutation (see Examples 1-2) andseparate spacer sequences designed to target the R838S mutation (seeExamples 13-14). For example, in the first grouping of Table 8, SEQ IDNOs: 5284 and 5304 are shown. SEQ ID NO: 5284 targets the R838H mutationand SEQ ID NO: 5304 targets the R838S mutation. An R838SH doublemutation spacer sequence can be designed by manipulating the bases shownin bold typeface. For example, a R838SH double mutation sgRNA, such asSEQ ID NO: 5437, was designed by replacing the bolded C residue in SEQID NO: 5284 with the bolded A residue in SEQ ID NO: 5304 to yield thebolded AA sequence. This same R838SH double mutation sgRNA could also bedesigned by starting with SEQ ID NO: 5304 and replacing the bolded Gresidue with an A residue of SEQ ID NO: 5284 to yield the bolded AAsequence. SEQ ID NOs: 5284, 5304, and 5437 are all 20mer spacersequences.

A similar process was used to generate the 19mer R838SH double mutationspacer sequence identified as SEQ ID NO: 5440, starting from SEQ ID NOs:5289 and 5309.

A similar process was used to generate the sgRNAs in the remaininggroupings in Table 8.

SEQ ID NOs: 5274 and 5278 (which bind to the wild-type GUCY2D allelewith zero mismatches) are also shown in the first grouping of Table 8 asa reference to the wild-type GUCY2D sequence.

SEQ ID NOs: 5436-5443 refer to sgRNA spacer sequences of R838SH doublemutation sgRNAs that associate with SpCas9. SEQ ID NOs: 5434-5435 referto sgRNA spacer sequences of R838SH double mutation sgRNAs thatassociate with SaCas9.

The R838SH double mutation gRNAs of the present disclosure can allow formore specific editing of the R838S and/or R838H mutant alleles, whilereducing off-target editing of a wild-type allele.

Example 21—Bioinformatics Analysis of the Guide RNAs

A gRNA or sgRNA can direct an RNP complex to an on-target site such as agenomic sequence for which editing is desired but may also have thepotential to interact with an off-target site for which editing is notdesired. To identify candidate gRNAs or sgRNAs that were likely to haveon-target and/or off-target activity, candidate gRNAs were screened andselected in a single process or multi-step process that used both insilico analysis of binding and experimentally assessed activity at bothon-target and off-target sites.

By way of illustration, candidate gRNAs having sequences that match aparticular on-target site, such as a site within or near the R838H,R838C, or R838S mutation in the GUCY2D gene, with an adjacent PAM wereassessed for their potential to cleave at off-target sites havingsimilar sequences, using one or more of a variety of bioinformaticstools. Such tools for assessing off-target binding are known in the art,examples of which are described and illustrated in more detail below.

Candidates predicted to have relatively lower potential for off-targetactivity were then assessed in in vitro experiments to measure theiron-target activity and off-target activities at various sites. GuideRNAs having sufficiently high on-target activity to achieve desiredlevels of gene editing at the selected locus, and relatively loweroff-target activity to reduce the likelihood of alterations at otherchromosomal loci are useful for modifying mutant GUCY2D. The ratio ofon-target to off-target activity is often referred to as the“specificity” of a guide.

For initial screening of predicted off-target activities, bioinformaticstools known and publicly available were used to predict the most likelyoff-target sites. Because binding to target sites in theCRISPR/Cas9/Cpf1 nuclease system is driven by Watson-Crick base pairingbetween complementary sequences, the degree of dissimilarity (andtherefore reduced potential for off-target binding) was related toprimary sequence differences: mismatches and bulges, i.e. bases thatwere changed to a non-complementary base, and insertions or deletions ofbases in the potential off-target site relative to the target site. Anexemplary bioinformatics tool called COSMID (CRISPR Off-target Siteswith Mismatches, Insertions and Deletions) (available atcrispr.bme.gatech.edu) compiles such similarities. Other bioinformaticstools include, but are not limited to autoCOSMID and CCTop.

Bioinformatics tools were used to minimize off-target cleavage in orderto reduce the detrimental effects of mutations and chromosomalrearrangements. Studies on CRISPR/Cas9 systems suggested the possibilityof off-target activity due to non-specific hybridization of the guidestrand to DNA sequences with base pair mismatches and/or bulges,particularly at positions distal from the PAM region. Therefore, it wasimportant to have a bioinformatics tool that identified potentialoff-target sites that have insertions and/or deletions between the RNAguide strand and genomic sequences, in addition to base-pair mismatches.Bioinformatics tools based upon the off-target prediction algorithmCCTop were used to search genomes for potential CRISPR off-target sites(CCTop is available on the web at crispr.cos.uni-heidelberg.de/). Theoutput ranked lists of the potential off-target sites based on thenumber and location of mismatches, allowing more informed choice oftarget sites, and avoiding the use of sites with more likely off-targetcleavage.

Additional bioinformatics pipelines were employed that weigh theestimated on- and/or off-target activity of gRNA targeting sites in aregion. Other features that were used to predict activity includeinformation about the cell type in question, DNA accessibility,chromatin state, transcription factor binding sites, transcriptionfactor binding data, and other CHIP-seq data. Additional factors wereweighed that predict editing efficiency, such as relative positions anddirections of pairs of gRNAs, local sequence features andmicro-homologies.

These processes allow for selection of high specificity gRNAs or sgRNAsfor further development.

Example 22—Testing of Guide RNAs in Cells for Off-Target Activity

To further evaluate the specificity of gRNAs provided herein, selectedgRNAs predicted to have the lowest off-target activity were tested foroff-target editing efficiency.

HEK 293FT cells with SpCas9 open reading frame (ORF) regulated by aconstitutive promoter integrated into the AAV51 locus were cultured in10% heat inactivated (HI) FBS/DMEM supplemented with 1 μg/ml puromycin,and passaged every 3-4 days.

The HEK 293FT cell line expressing SpCas9 was seeded in 100 μl of 10%HI-FBS/DMEM at 50,000 cells per well in a 96-well plate, and transfectedwith 1 μg of sgRNA using Lipofectamine® MessengerMax™ (available fromThermo Fisher Scientific, Massachusetts, US). sgRNAs used for this assaywere synthesized by in vitro transcription (IVT). The DNA fragmentscontaining bacteriophage T7 promoter, protospacer and sgRNA tracersequences were generated by assembling oligonucleotides through PCR. IVTwas performed using the TranscriptAid T7 High Yield Transcription Kit(available from Thermo Fisher Scientific), and the synthesized RNAstrands were purified using either ZR-96 RNA Clean & Concentrator™(available from Zymo Research, California, US) or RNAClean XP beads(available from Beckman Coulter, California, US).

At 48 hours post-transfection, medium was removed and total DNA wasextracted using prepGem® Tissue Kit (available from VWR, Pennsylvania,US). The sequence surrounding the Cas9 target sites on the genome wasPCR-amplified. The Cas9 target sites on the genome were the wild-typeGUCY2D locus, the R838H mutation, the R838C mutation, or the R838Smutation. The resulting products were cleaned up using AMPure XP beads(available from Beckman Coulter), and sequenced to assess Cas9-mediatedgenetic modifications. The frequencies of small insertions and deletions(indels) were estimated using TIDE.

On-target editing efficiency was determined at the wild-type GUCY2Dlocus via TIDE analysis for sgRNAs that target the wild-type GUCY2D gene(FIGS. 4A-D; sgRNAs comprising SEQ ID NOs: 5274, 5278, 5275, 5279, 5276,5280, 5277, or 5281). These sgRNAs that target the wild-type GUCY2Dlocus were used as positive controls to measure on-target editing at thewild-type GUCY2D locus.

Off-target editing efficiency was determined at the wild-type GUCY2Dlocus via TIDE analysis for sgRNAs that target the R838H mutation (FIGS.4A-E; sgRNAs comprising SEQ ID NOs: 5284, 5289, 5285, 5290, 5286, 5291,5287, 5292, 5288, or 5293).

Off-target editing efficiency was also determined at the wild-typeGUCY2D locus via TIDE analysis for sgRNAs that target the R838C mutation(FIGS. 4A-D; sgRNAs comprising SEQ ID NOs: 5296, 5300, 5297, 5301, 5298,5302, 5299, or 5303).

Off-target editing efficiency was also determined at the wild-typeGUCY2D locus via TIDE analysis for sgRNAs that target the R838S mutation(FIGS. 4A-D; sgRNAs comprising SEQ ID NOs: 5304, 5308, 5305, 5309, 5306,5310, 5307, or 5311).

Off-target editing efficiency was also determined at the wild-typeGUCY2D locus via TIDE analysis for sgRNAs that are able to target boththe R838H mutation and R838C mutation (FIGS. 4A-E; sgRNAs comprising SEQID NOs: 5398, 5403, 5399, 5404, 5400, 5405, 5402, 5407, 5401, or 5406).

These data provide evidence that the selected gRNAs can minimizeoff-target activity (FIGS. 4A-E).

Example 23—Testing of Guide RNAs in Cells for On-Target Activity

To further evaluate the specificity of gRNAs provided herein, selectedgRNAs predicted to have the lowest off-target activity were also testedfor on-target editing efficiency.

HEK 293FT cells expressing SpCas9 were collected and resuspended in SFsolution at 10,000,000 cells per ml. 20 μl of the cell suspension wastransfected with 2 μg of sgRNA and 200, 400 or 800 ng of either aplasmid encoding the wild-type GUCY2D gene or a plasmid encoding theR838H mutation. The transfection of the HEK 293 FT cells was performedby nucleofection under the program CM-130 using Lonza 4D nucleofectorsystem.

sgRNAs used for this assay were synthesized by IVT. The DNA fragmentscontaining bacteriophage T7 promoter, protospacer and sgRNA tracersequences were generated by assembling oligonucleotides through PCR. IVTwas performed using TranscriptAid T7 High Yield Transcription Kit, andthe synthesized RNA strands were purified using either ZR-96 RNA Clean &Concentrator™ or RNA clean XP beads (Beckman Coulter).

A plasmid encoding the wild-type GUCY2D gene or a plasmid encodingGUCY2D comprising the R838H mutation was built by two rounds ofmolecular cloning. First, pSpCas9 (purchased from Genscript, New Jersey,US) was digested with KpnI-HF and EcoRI-HF, and a PCR fragmentcontaining human rhodopsin kinase promoter (GRK1) and the sequenceencoding the amino-acid residue 962 through the C-terminus of humanGUCY2D was inserted between the two digestion sites.

The resulting plasmid and ORF cDNA clones encoding the wild type GUCY2D(purchased from Genecopoeia) were digested with BstBI and NdeI, and thelinearized fragments were ligated with T4 DNA ligase to insert thefull-length wild-type GUCY2D gene downstream of the GRK1 promoter.

In addition, the resulting plasmid and ORF cDNA clones encoding theR838H mutation (purchased from Genecopoeia) were digested with BstBI andNdeI, and the linearized fragments were ligated with T4 DNA ligase toinsert the full-length R838H containing GUCY2D gene downstream of theGRK1 promoter.

Each 25 μl of the nucleofection samples was transferred into 1-wellcontaining 175 μl of 10% HI-FBS/DMEM in 96-well plates. At 48-hourspost-transfection, each well was washed with PBS twice, and total DNAwas extracted using prepGem® Tissue Kit. The sequence surrounding Cas9target sites on the plasmid DNA was PCR-amplified. The resultingproducts were cleaned up using AMPure XP beads, and sequenced to assessCas9-mediated genetic modifications. The frequencies of small insertionsand deletions (indels) were estimated using TIDE.

On-target editing efficiency was determined at the wild-type GUCY2Dlocus via TIDE analysis for sgRNAs that target the wild-type GUCY2D gene(FIG. 5; sgRNAs comprising SEQ ID NO: 5274).

On-target editing efficiency was also determined at the R838H mutationof the GUCY2D gene via TIDE analysis for sgRNAs that target the R838Hmutation (FIG. 5; sgRNAs comprising SEQ ID NO: 5284, 5285, 5286, 5287,or 5288).

On-target editing efficiency was also determined at the R838H mutationof the GUCY2D gene via TIDE analysis for sgRNAs that are able to targetboth the R838H mutation and R838C mutation (FIG. 5; sgRNAs comprisingSEQ ID NO: 5398, 5399, 5400, 5401, or 5402).

These data provide evidence that selected gRNAs designed by Applicantscan effectively edit a GUCY2D gene harboring a R838H mutation (FIG. 5).

Example 24—Testing of Guide RNAs in Cells for On-Target and Off-TargetActivity

To further evaluate the specificity of gRNAs provided herein, selectedgRNAs were further tested for on-target and off-target activity.

A sequence containing human U6 promoter, seamless protospacer cloningsite, sgRNA tracer, CMV promoter, chimeric intron, EGFP ORF, WPRE andSV40 polyadenylation signal was synthesized using GeneArt service(ThermoFisher Scientific). This sequence was separated from the vectorbackbone by digestion with MluI-HF and RsrII, and cloned between thesame sites on the AAV vector. The resulting plasmid (pSIA003) wasdigested with SapI, and synthesized oligonucleotides were annealed togenerate protospacers with 3-nt overhangs compatible with those of thelinearized pSIA003. The double-stranded protospacers were inserted intopSIA003 by DNA ligation.

HEK 293FT SpCas9-expressing cells were transfected with 200, 400 or 800ng of pSIA003, which contains a U6-driven sgRNA. HEK 293FT SpCas9expressing cell samples were referenced in FIGS. 6A-B as 2, 4, and 8 intheir sample names depending on the concentration of pSIA003 that wasused (200, 400, or 800 ng). At the same time that HEK 293FTSpCas9-expressing cells were transfected with pSIA003, they were alsotransfected with 200 ng of a plasmid encoding the wild-type GUCY2D gene,a plasmid encoding GUCY2D comprising the R838H mutation, or a plasmidencoding GUCY2D comprising the R838C mutation. The transfection of theHEK 293FT cells was performed by nucleofection under the program CM-130using a Lonza 4D-Nucleofector™ system (available from Lonza,Switzerland).

Each 25 μl of the nucleofection samples was transferred into 1-wellcontaining 175 μl of 10% HI-FBS/DMEM in 96-well plates. At 48 and 72hours post-transfection, each well was washed with PBS twice, then totalDNA was extracted using prepGem Tissue Kit. The sequence surroundingCas9 target sites on the plasmid DNA was PCR-amplified. The resultingproducts were cleaned up using AMPure XP beads, and sequenced to assessCas9-mediated genetic modifications. The frequencies of small insertionsand deletions (indels) were estimated using TIDE.

On-target editing efficiency was determined at the wild-type GUCY2Dlocus via TIDE analysis for a sgRNA that targets the wild-type GUCY2Dgene (sgRNA comprising SEQ ID NO: 5274) at 48 hours (FIG. 6A) and 72hours (FIG. 6B) post-transfection.

On-target and off-target editing efficiency was also determined at thewild-type GUCY2D locus, the R838H mutation of the GUCY2D gene, and theR838C mutation of the GUCY2D gene via TIDE analysis for sgRNAs thattarget the R838H mutation (sgRNAs comprising SEQ ID NOs: 5285 or 5286)at 48 hours (FIG. 6A) and 72 hours (FIG. 6B) post-transfection.

On-target and off-target editing efficiency was also determined at thewild-type GUCY2D locus, the R838H mutation of the GUCY2D gene, and theR838C mutation of the GUCY2D gene via TIDE analysis for sgRNAs that areable to target both the R838H mutation and R838C mutation (sgRNAscomprising SEQ ID NOs: 5398, 5399, or 5402) at 48 hours (FIG. 6A) and 72hours (FIG. 6B) post-transfection.

These data provide evidence that the selected gRNAs can effectively editthe GUCY2D mutations R838C and R838H while minimizing off-targetactivity (FIGS. 6A-B).

Example 25—Testing of Guide RNAs in Cells for On-Target and Off-TargetActivity

To further evaluate the specificity of gRNAs provided herein, selectedgRNAs were further tested for on-target and off-target activity whentargeting a genomic GUCY2D allele for editing.

Three reporter cell lines were generated that contain a Cas9 target sitefused to a blue fluorescent protein at the beta-tubulin gene locus. Thefirst reporter cell line has a wild-type GUCY2D gene (Cas9 target site)fused to a blue fluorescent protein at the beta-tubulin gene locus. Thesecond reporter cell line has a GUCY2D gene comprising a R838H mutation(Cas9 target site) fused to a blue fluorescent protein at thebeta-tubulin gene locus. The third reporter cell line has a GUCY2D genecomprising a R838C mutation (Cas9 target site) fused to a bluefluorescent protein at the beta-tubulin gene locus.

These three reporter cell lines were generated using pDL124 and a donorplasmid. pDL124 contains a SpCas9 ORF under CMV promoter and a U6promoter-driven sgRNA and was built by Gibson assembly. The U6promoter-driven sgRNA can cut the codon corresponding to the thirdresidue from the C-terminus of beta-tubulin. A donor plasmid (FIG. 7A)comprises a Cas9 target site (either a wild-type GUCY2D gene, a GUCY2Dgene comprising a R838H mutation, or a GUCY2D gene comprising a R838Cmutation). Each of these 3 donor plasmids was synthesized using GeneArtservice (ThermoFisher Scientific).

HEK 293FT cells (200,000 cells) were transfected with 0.5 μg of pDL124and 0.5 μg of donor plasmid by nucleofection under the program DN-100.pDL124 was used to generate a double-strand break at the beta-tubulingene locus of the HEK 293FT cells in order to integrate the Cas9 targetsite (i.e. the wild-type GUCY2D gene, the GUCY2D gene comprising theR838H mutation, or the GUCY2D gene comprising the R838C mutation) fromthe donor plasmid and into the beta-tubulin gene locus of the HEK 293FTcells between the two homologous arms (tubb exon 4). Homologousrecombination leads to expression of beta-tubulin fused to T2A peptide.The Cas9 target site includes no stop codon, and blue fluorescentprotein (mTagBFP2) and blasticidin selection marker (bsd) are encoded inthe same reading frame as for beta-tubulin. Cells with a Cas9 targetsite integrated correctly were enriched in 10% FBS/DMEM supplementedwith 2-5 μg/ml blasticidin, and isolated by cell sorting. Each of thereporter cell lines was seeded in 2.5 ml of 10% FBS/DMEM at 500,000cells per well in 6-well plates 24 hours before transfection.

The reporter cell line having a wild-type GUCY2D gene (Cas9 target site)fused to a blue fluorescent protein at the beta-tubulin gene locus wastransfected with 1.25 μg pSIA043 and 1.25 μg pSIA012 usingLipofectamine® 3000 (FIG. 7B). pSIA043 encodes Cas9. A nucleotidesequence containing GRK1-driven SpCas9 and SV40 polyadenylation signalwas synthesized using GeneArt service (ThermoFisher Scientific), andtransferred onto the AAV vector by conventional DNA cloning techniques.The human elongation factor 1 alpha promoter was PCR-amplified, andsubstituted for GRK1 promoter. The resulting plasmid was designated aspSIA043. pSIA012 is pAAV-5285, which comprises an AAV sequence thatencodes for R838H_Sp_T2 sgRNA (a sgRNA comprising SEQ ID NO: 5285) andEGFP (SEQ ID NO: 5469).

The reporter cell line having a GUCY2D gene comprising a R838H mutation(Cas9 target site) fused to a blue fluorescent protein at thebeta-tubulin gene locus was transfected with 1.25 μg pSIA043 and 1.25 μgpSIA012 using Lipofectamine® 3000 (FIG. 7E).

The reporter cell line having a GUCY2D gene comprising a R838C mutation(Cas9 target site) fused to a blue fluorescent protein at thebeta-tubulin gene locus was transfected with 1.25 μg pSIA043 and 1.25 μgpSIA012 using Lipofectamine® 3000 (FIG. 7H).

The reporter cell line having a wild-type GUCY2D gene (Cas9 target site)fused to a blue fluorescent protein at the beta-tubulin gene locus wastransfected with 1.25 μg pSIA043 and 1.25 μg pSIA015 usingLipofectamine® 3000 (FIG. 7C). pSIA015 is pAAV-5398, a pAAV comprising asequence that encodes for R838CH_Sp_T1 sgRNA (a sgRNA comprising SEQ IDNO: 5398) and EGFP (SEQ ID NO: 5471).

The reporter cell line having a GUCY2D gene comprising a R838H mutation(Cas9 target site) fused to a blue fluorescent protein at thebeta-tubulin gene locus was transfected with 1.25 μg pSIA043 and 1.25 μgpSIA015 using Lipofectamine® 3000 (FIG. 7F).

The reporter cell line having a GUCY2D gene comprising a R838C mutation(Cas9 target site) fused to a blue fluorescent protein at thebeta-tubulin gene locus was transfected with 1.25 μg pSIA043 and 1.25 μgpSIA015 using Lipofectamine® 3000 (FIG. 7I).

The reporter cell line having a wild-type GUCY2D gene (Cas9 target site)fused to a blue fluorescent protein at the beta-tubulin gene locus wastransfected with only transfection reagent (no DNA) using Lipofectamine®3000 (FIG. 7D).

The reporter cell line having a GUCY2D gene comprising a R838H mutation(Cas9 target site) fused to a blue fluorescent protein at thebeta-tubulin gene locus was transfected with only transfection reagent(no DNA) using Lipofectamine® 3000 (FIG. 7G).

The reporter cell line having a GUCY2D gene comprising a R838C mutation(Cas9 target site) fused to a blue fluorescent protein at thebeta-tubulin gene locus was transfected with only transfection reagent(no DNA) using Lipofectamine® 3000 (FIG. 7J).

At 72 hours post-transfection, reporter cells were dissociated from theplates by incubation with trypsin-EDTA, and analyzed for bluefluorescence (BFP) and green fluorescence (GFP) by flow cytometry. Aframe-shift induced by genome editing at a Cas9 target site (whether theCas9 target site is the wild-type GUCY2D gene, the GUCY2D genecomprising the R838H mutation, or the GUCY2D gene comprising the R838Cmutation) of the HEK 293FT cell results in loss of BFP. EGFP and sgRNAare encoded on the same vector and the EGFP serves as a transfectionmarker. Therefore, HEK 293FT cells transfected with pSIA012 (whichcomprises a sequence that encodes R838H_Sp_T2 sgRNA—a sgRNA comprisingSEQ ID NO: 5285) or pSIA015 (which comprises a sequence that encodesR838CH_Sp_T1 sgRNA—a sgRNA comprising SEQ ID NO: 5398) are GFP positive.

Gene editing in the transfected cells was estimated in the cellpopulations plotted within gate “B” (FIGS. 7B-7J). The boldedpercentages in FIGS. 7B-7J indicate BFP negative and GFP positive cellswithin gate “B”. BFP negative means that gene editing occurred at theCas9 target site of these transfected HEK 293FT cells. GFP positivemeans that these transfected HEK 293FT cells contain a plasmid thatencodes EGFP and sgRNA.

FIG. 7B shows that of the transfected HEK 293FT reporter cells that havethe wild-type GUCY2D gene as the Cas9 target site, 16.67% of these cellshad the wild-type GUCY2D gene edited when R838H_Sp_T2 sgRNA (sgRNAcomprising SEQ ID NO: 5285) was used as the sgRNA.

FIG. 7C shows that of the transfected HEK 293FT reporter cells that havethe wild-type GUCY2D gene as the Cas9 target site, 3.38% of these cellshad the wild-type GUCY2D gene edited when R838CH_Sp_T1 sgRNA (sgRNAcomprising SEQ ID NO: 5398) was used as the sgRNA.

FIG. 7D shows that of the transfected HEK 293FT reporter cells that havethe wild-type GUCY2D gene as the Cas9 target site, 0% of these cells hadthe wild-type GUCY2D gene edited when no sgRNA was used.

FIG. 7E shows that of the transfected HEK 293FT reporter cells that havethe R838H mutation as the Cas9 target site, 52.37% of these cells hadthe R838H mutation edited when R838H_Sp_T2 sgRNA (sgRNA comprising SEQID NO: 5285) was used as the sgRNA.

FIG. 7F shows that of the transfected HEK 293FT reporter cells that havethe R838H mutation as the Cas9 target site, 16.14% of these cells hadthe R838H mutation edited when R838CH_Sp_T1 sgRNA (sgRNA comprising SEQID NO: 5398) was used as the sgRNA.

FIG. 7G shows that of the transfected HEK 293FT reporter cells that havethe R838H mutation as the Cas9 target site, 0% of these cells had theR838H mutation edited when no sgRNA was used.

FIG. 7H shows that of the transfected HEK 293FT reporter cells that havethe R838C mutation as the Cas9 target site, 1.89% of these cells had theR838C mutation edited when R838H_Sp_T2 sgRNA (sgRNA comprising SEQ IDNO: 5285) was used as the sgRNA.

FIG. 7I shows that of the transfected HEK 293FT reporter cells that havethe R838C mutation as the Cas9 target site, 24.47% of these cells hadthe R838C mutation edited when R838CH_Sp_T1 sgRNA (sgRNA comprising SEQID NO: 5398) was used as the sgRNA.

FIG. 7J shows that of the transfected HEK 293FT reporter cells that havethe R838C mutation as the Cas9 target site, 0% of these cells had theR838C mutation edited when no sgRNA was used.

Because no genome editing was induced in mock transfected cells, thevast majority of mock transfected cells are BFP positive (FIGS. 7D, 7G,and 7J).

Genomic DNA was also extracted from reporter cells 72-hourspost-transfection using Zymo Research Quick-DNA plus kit. The sequencespanning Cas9 target sites upstream of the fluorescent protein gene wasPCR-amplified, and indels were analyzed by TIDE (data not shown). Theresults of those data not shown were in agreement with the resultsobserved by flow cytometry analysis.

These data provide evidence that the tested gRNAs can effectively editthe mutant R838H GUCY2D gene and R838C GUCY2D gene while minimizingoff-target activity (FIGS. 7B-J).

Example 26—cGMP Functional Assay

The GUCY2D protein is a guanylate cyclase, which synthesizes cGMP inmammalian photoreceptor cells. A cGMP functional assay was establishedas an on-target screening approach for R838H sgRNA screening. In theassay, in vitro-transcribed (IVT) sgRNAs and a vector containing R838HcDNA were co-transfected into HEK293T-SpCas9 cells. Editing of the R838HcDNA can result in reduction of GUCY2D protein production, andconsequently decreased cGMP signal. Assays were also conducted usingAAV-encoded sgRNAs. The editing efficiency of gRNAs was directlyproportional to the percent reduction of cGMP in cells transfected withsgRNAs.

A total of 10 sgRNAs targeting the R838H mutation within the GUCY2D gene(sgRNAs comprising SEQ ID NOs: 5284, 5285, 5286, 5287, 5288, 5289, 5290,5291, 5292, or 5293) were screened for on-target editing using the cGMPfunctional assay. To calculate percent reduction of cGMP (FIG. 8A),HEK293T-SpCas9 cells co-transfected with (1) a R838H IVT sgRNA and (2) avector containing R838H cDNA were compared to HEK293T-SpCas9 cells thatwere not transfected with any sgRNA but did receive the cDNA vector. Forexample, HEK293T-SpCas9 cells co-transfected with a vector containingR838H cDNA and sgRNA comprising SEQ ID NO: 5284 had a 69.1% reduction ofcGMP, which demonstrates that the sgRNA comprising SEQ ID NO: 5284 wasable to edit the R838H mutation within the GUCY2D gene. HEK293T-SpCas9cells co-transfected with a vector containing R838H cDNA and sgRNAcomprising SEQ ID NO: 5285 had a 72.5% reduction of cGMP, whichdemonstrates that the sgRNA comprising SEQ ID NO: 5285 was able to editthe R838H mutation within the GUCY2D gene. HEK293T-SpCas9 cellsco-transfected with a vector containing R838H cDNA and sgRNA comprisingSEQ ID NO: 5286 had a 51.9% reduction of cGMP, which demonstrates thatthe sgRNA comprising SEQ ID NO: 5286 was able to edit the R838H mutationwithin the GUCY2D gene. HEK293T-SpCas9 cells co-transfected with avector containing R838H cDNA and sgRNA comprising SEQ ID NO: 5287 had a25.9% reduction of cGMP, which demonstrates that the sgRNA comprisingSEQ ID NO: 5287 was able to edit the R838H mutation within the GUCY2Dgene. HEK293T-SpCas9 cells co-transfected with a vector containing R838HcDNA and sgRNA comprising SEQ ID NO: 5288 had a 28.1% reduction of cGMP,which demonstrates that the sgRNA comprising SEQ ID NO: 5288 was able toedit the R838H mutation within the GUCY2D gene. HEK293T-SpCas9 cellsco-transfected with a vector containing R838H cDNA and sgRNA comprisingSEQ ID NO: 5289 had a 73.1% reduction of cGMP, which demonstrates thatthe sgRNA comprising SEQ ID NO: 5289 was able to edit the R838H mutationwithin the GUCY2D gene. HEK293T-SpCas9 cells co-transfected with avector containing R838H cDNA and sgRNA comprising SEQ ID NO: 5290 had a70.3% reduction of cGMP, which demonstrates that the sgRNA comprisingSEQ ID NO: 5290 was able to edit the R838H mutation within the GUCY2Dgene. HEK293T-SpCas9 cells co-transfected with a vector containing R838HcDNA and sgRNA comprising SEQ ID NO: 5291 had a 60.1% reduction of cGMP,which demonstrates that the sgRNA comprising SEQ ID NO: 5291 was able toedit the R838H mutation within the GUCY2D gene. HEK293T-SpCas9 cellsco-transfected with a vector containing R838H cDNA and sgRNA comprisingSEQ ID NO: 5292 had a 47.8% reduction of cGMP, which demonstrates thatthe sgRNA comprising SEQ ID NO: 5292 was able to edit the R838H mutationwithin the GUCY2D gene. HEK293T-SpCas9 cells co-transfected with avector containing R838H cDNA and sgRNA comprising SEQ ID NO: 5293 had a20.9% reduction of cGMP, which demonstrates that the sgRNA comprisingSEQ ID NO: 5293 was able to edit the R838H mutation within the GUCY2Dgene. Absolute cGMP was also determined for these same samples (FIG.8B).

A sgRNA comprising SEQ ID NO: 5277 that targets the wild-type GUCY2Dgene and a vector containing wild-type GUCY2D cDNA were co-transfectedinto HEK293T-SpCas9 cells and used as a control. Editing of wild-typeGUCY2D cDNA using the sgRNA that targets the wild-type GUCY2D generesulted in an 80% reduction of cGMP (data not shown), whichdemonstrates that the sgRNA comprising SEQ ID NO: 5277 was able to editthe wild-type GUCY2D. Percent reduction of cGMP was calculated bycomparing the cGMP levels of cells transfected with the sgRNA comprisingSEQ ID NO: 5277 that targets the wild-type GUCY2D gene compared to cellsthat were not transfected with any sgRNAs (data not shown). Editing ofwild-type GUCY2D cDNA using the sgRNA that targets the wild-type GUCY2Dgene resulted in ˜225 nM cGMP (data not shown).

In a second cGMP functional assay, an IVT sgRNA and a vector containingR838H cDNA were co-transfected into HEK293T-SpCas9 cells. Three sgRNAsthat target the R838H mutation within the GUCY2D gene (sgRNAs comprisingSEQ ID NOs: 5285, 5286 or 5291) were screened for on-target editingusing the cGMP functional assay. To calculate percent reduction of cGMP(FIG. 9A), HEK293T-SpCas9 cells co-transfected with (1) a R838H IVTsgRNA and (2) a vector containing R838H cDNA were compared toHEK293T-SpCas9 cells that were not transfected with any sgRNA but didreceive the cDNA vector. For example, HEK293T-SpCas9 cellsco-transfected with a vector containing R838H cDNA and sgRNA comprisingSEQ ID NO: 5285 had an 82.7% reduction of cGMP, which demonstrates thatthe sgRNA comprising SEQ ID NO: 5285 was able to edit the R838H mutationwithin the GUCY2D gene. HEK293T-SpCas9 cells co-transfected with avector containing R838H cDNA and sgRNA comprising SEQ ID NO: 5286 had a60.5% reduction of cGMP, which demonstrates that the sgRNA comprisingSEQ ID NO: 5286 was able to edit the R838H mutation within the GUCY2Dgene. HEK293T-SpCas9 cells co-transfected with a vector containing R838HcDNA and sgRNA comprising SEQ ID NO: 5291 had a 69.1% reduction of cGMP,which demonstrates that the sgRNA comprising SEQ ID NO: 5291 was able toedit the R838H mutation within the GUCY2D gene. Absolute cGMP was alsodetermined for these same samples (FIG. 9B)

In a third cGMP functional assay, an AAV vector encoding a sgRNA drivenby a U6 promoter (pAAV-U6-sgRNA) and a vector containing R838H cDNA wereco-transfected into HEK293T-SpCas9 cells. sgRNAs that target the R838Hmutation within the GUCY2D gene (sgRNAs comprising SEQ ID NOs: 5285 or5286) were screened for on-target editing using the cGMP functionalassay. Percent reduction of cGMP (FIG. 10A) was calculated by comparingthe cGMP level of HEK293T-SpCas9 cells co-transfected with apAAV-U6-R838H sgRNA and a vector containing R838H cDNA withHEK293T-SpCas9 cells that were not transfected with any pAAV-U6-R838HsgRNA. For example, HEK293T-SpCas9 cells co-transfected with a vectorcontaining R838H cDNA and pSIA012 (also called pAAV-5285), a plasmidcomprising an AAV sequence that encodes for a sgRNA comprising SEQ IDNO: 5285) had a 41.3% reduction of cGMP, which demonstrates that thesgRNA comprising SEQ ID NO: 5285 was able to edit the R838H mutationwithin the GUCY2D gene. HEK293T-SpCas9 cells co-transfected with avector containing R838H cDNA and pAAV-U6-R838H sgRNA (also calledpAAV-5286, a plasmid comprising an AAV sequence that encodes for a sgRNAcomprising SEQ ID NO: 5286) had a 16.5% reduction of cGMP, whichdemonstrates that the sgRNA comprising SEQ ID NO: 5286 was able to editthe R838H mutation within the GUCY2D gene. HEK293T-SpCas9 cellsco-transfected with a vector containing R838H cDNA and pAAV-U6-WT sgRNA(a pAAV-5274, a plasmid comprising an AAV sequence that encodes for asgRNA comprising SEQ ID NO: 5274) had a 0% reduction of cGMP, whichdemonstrates that the sgRNA comprising SEQ ID NO: 5274 was not able toedit the R838H mutation within the GUCY2D gene. Absolute cGMP was alsodetermined for these same samples (FIG. 10B)

An AAV vector encoding a sgRNA driven by a U6 promoter that targets thewild-type GUCY2D gene (pAAV-5274, a plasmid comprising an AAV sequencethat encodes for a sgRNA comprising SEQ ID NO: 5274) and a vectorcontaining wild-type GUCY2D cDNA were co-transfected into HEK293T-SpCas9cells and used as a control. Editing of wild-type GUCY2D cDNA using thesgRNA comprising SEQ ID NO: 5274 that targets the wild-type GUCY2D generesulted in a 42.2% reduction of cGMP (data not shown), whichdemonstrates that the sgRNA comprising SEQ ID NO: 5274 was able to editthe wild-type GUCY2D. Percent reduction of cGMP was calculated bycomparing the cGMP level of HEK293T-SpCas9 cells co-transfected with asgRNA comprising SEQ ID NO: 5274 that targets the wild-type GUCY2D genecompared to cells that were not transfected with any sgRNAs (data notshown). Editing of wild-type GUCY2D cDNA using pAAV comprising SEQ IDNO: 5274 resulted in ˜325 nM cGMP (data not shown).

These data provide evidence that the tested gRNAs can effectively edit aGUCY2D gene containing the R838H mutation regardless of whether thegRNAs are synthetic gRNAs (FIGS. 8A-B and 9A-B), or AAV-encoded gRNAs(FIGS. 10A-B). Additionally, these data provide evidence that gRNAs ofthe present disclosure can reduce protein expression from the GUCY2Dgene in support of at least the NHEJ editing strategy (FIGS. 8-10).

Example 27—Testing of Guide RNAs in Cells for Off-Target Activity

To determine the extent of off-target editing on a genomic level, thegRNAs (or sgRNAs) having the best on-target activity will then be testedfor targeted-genome-wide off-target editing using GUIDE-seq,Amplicon-seq, and/or Digenome-seq. Off-target effects will be tested inhuman cells. This testing can enable selection of gRNAs with increasedspecificity.

Example 28—Testing Different Approaches for HDR Gene Editing

After testing the gRNAs for both on-target activity and off-targetactivity, mutation correction and knock-in strategies will be tested forHDR gene editing.

For the mutation correction approach, donor DNA template will beprovided as a short single-stranded oligonucleotide, a shortdouble-stranded oligonucleotide (PAM sequence intact/PAM sequencemutated), a long single-stranded DNA molecule (PAM sequence intact/PAMsequence mutated) or a long double-stranded DNA molecule (PAM sequenceintact/PAM sequence mutated). In addition, the donor DNA template willbe delivered by AAV.

For the cDNA knock-in approach, a single-stranded or double-stranded DNAhaving homologous arms to the GUCY2D chromosomal region can include morethan 40 nt of the first exon (the first coding exon) of the GUCY2D gene,the complete CDS of the GUCY2D gene and 3′ UTR of the GUCY2D gene, andat least 40 nt of the following intron. The single-stranded ordouble-stranded DNA having homologous arms to the GUCY2D chromosomalregion can include more than 80 nt of the first exon of the GUCY2D gene,the complete CDS of the GUCY2D gene and 3′ UTR of the GUCY2D gene, andat least 80 nt of the following intron. The single-stranded ordouble-stranded DNA having homologous arms to the GUCY2D chromosomalregion can include more than 100 nt of the first exon of the GUCY2Dgene, the complete CDS of the GUCY2D gene and 3′ UTR of the GUCY2D gene,and at least 100 nt of the following intron. The single-stranded ordouble-stranded DNA having homologous arms to the GUCY2D chromosomalregion can include more than 150 nt of the first exon of the GUCY2Dgene, the complete CDS of the GUCY2D gene and 3′ UTR of the GUCY2D gene,and at least 150 nt of the following intron. The single-stranded ordouble-stranded DNA having homologous arms to the GUCY2D chromosomalregion can include more than 300 nt of the first exon of the GUCY2Dgene, the complete CDS of the GUCY2D gene and 3′ UTR of the GUCY2D gene,and at least 300 nt of the following intron. The single-stranded ordouble-stranded DNA having homologous arms to the GUCY2D chromosomalregion can include more than 400 nt of the first exon of the GUCY2Dgene, the complete CDS of the GUCY2D gene and 3′ UTR of the GUCY2D gene,and at least 400 nt of the following intron.

Alternatively, the DNA template will be delivered by a recombinant AAVparticle such as those taught herein.

A knock-in of GUCY2D cDNA can be performed into any selected chromosomallocation or in one of the “safe harbor” locus, i.e., albumin gene, anAAV5 1 gene, an HRPT gene, a CCR5 gene, a globin gene, TTR gene, TFgene, F9 gene, Alb gene, Gys2 gene and PCSK9 gene. Assessment ofefficiency of HDR mediated knock-in of cDNA into the first exon canutilize cDNA knock-in into “safe harbor” sites such as: single-strandedor double-stranded DNA having homologous arms to one of the followingregions, for example: AAV51 19q13.4-qter, HRPT 1q31.2, CCR5 3p21.31,Globin 11p15.4, TTR 18q12.1, TF 3q22.1, F9 Xq27.1, Alb 4q13.3, Gys212p12.1, PCSK9 1p32.3; 5′UTR correspondent to GUCY2D or alternative 5′UTR, complete CDS of GUCY2D and 3′ UTR of GUCY2D or modified 3′ UTR andat least 80 nt of the first intron, alternatively same DNA templatesequence will be delivered by AAV.

These tests will allow for optimization of the various HDR gene editingstrategies and comparisons based on their respective effectiveness willbe made.

Example 29—Re-Assessment of Lead CRISPR-Cas9/DNA Donor Combinations

After testing the different strategies for gene editing, the leadCRISPR-Cas9/DNA donor combinations will be re-assessed in cells forefficiency of deletion, recombination, and off-target specificity. Cas9mRNA or RNP will be formulated into lipid nanoparticles for delivery,sgRNAs will be formulated into nanoparticles or delivered as arecombinant AAV particle, and donor DNA will be formulated intonanoparticles or delivered as recombinant AAV particle. These tests willallow for further optimization of the various HDR gene editingstrategies.

Example 30—Self-Inactivating (SIN) CRISPR-Cas Systems

When nucleic acids encoding Cas9 and/or guide RNA are delivered viaviral vector, it can be advantageous to use a SIN vector to deliver atleast one of the nucleic acids. Experiments were performed in order tofurther investigate the ability of various SIN vectors to edit targetednucleic acids with specificity.

The three reporter cell lines described in Example 25 were used. Thefirst reporter cell line has a wild-type GUCY2D gene (Cas9 target site)fused to a blue fluorescent protein (BFP) at the beta-tubulin genelocus. The second reporter cell line has a GUCY2D gene comprising aR838H mutation (Cas9 target site) fused to a BFP at the beta-tubulingene locus. The third reporter cell line has a GUCY2D gene comprising aR838C mutation (Cas9 target site) fused to a BFP at the beta-tubulingene locus. Thus, the Cas9 target site-BFP gene fusions comprised by thereporter cell lines can be used to report on editing activity at theCas9 target site. The editing activity can cause loss of the BFP signalvia a frameshift mutation. It was found that various combinations ofCas9 vectors and guide RNAs according to the present disclosure wereeffective in editing targeted GUCY2D R838H or R838C mutant Cas9 targetsites. The various combinations were also specific such that editing ofthe wild-type GUCY2D Cas9 target sites was minimal and similar tobackground levels of BFP signal loss.

Each of the reporter cell lines was seeded in 2.5 ml of 10% FBS/DMEM at500,000 cells per well in 6-well plates at 24 hours before transfection.

FIG. 12A shows results obtained when the reporter cell line having awild-type GUCY2D gene (Cas9 target site) fused to a blue fluorescentprotein at the beta-tubulin gene locus was transfected with 1.25 μgSIN-AAV SpCas9 ver. 1 (FIG. 11A) and 1.25 μg pSIA012 (FIG. 11D) usingLipofectamine® 3000. pSIA012 is a plasmid comprising an AAV sequence(SEQ ID NO: 5506) that encodes for R838H_Sp_T2 sgRNA (a sgRNA comprisingSEQ ID NO: 5285) and EGFP. SIN-AAV SpCas9 ver. 1 encodes SpCas9 andincludes SIN sites (also called R838 target sites) located 5′ (SEQ IDNO: 5478) and 3′ (SEQ ID NO: 5480) of the SpCas9 ORF. The 5′ SIN site(SEQ ID NO: 5478) in SIN-AAV SpCas9 ver. 1 comprises SEQ ID NO: 5327,which is targeted by sgRNA comprising SEQ ID NO: 5285. The 3′ SIN site(SEQ ID NO: 5480) in SIN-AAV SpCas9 ver. 1 comprises SEQ ID NO: 5369,which is also targeted by sgRNA comprising SEQ ID NO: 5285 (Table 9).

TABLE 9 SIN-AAV SpCas9 ver. 1 comprising two SIN sitestargeted by a sgRNA comprising SEQ ID NO: 5285 SEQ ID Sequence Type NO:Sequences 5′ SIN site sequence 5478 ggaggatctgatccgggagcacacggaggagctgga Target sequence 5327 ggatctgatccgggagcaca3′ SIN site sequence 5480 tccagctcctccgtgtgctc ccggatcagatcctccTarget sequence 5369 tgtgctcccggatcagatcc

The reporter cell line having a GUCY2D gene comprising a R838H mutation(Cas9 target site) fused to a blue fluorescent protein at thebeta-tubulin gene locus was transfected with 1.25 μg SIN-AAV SpCas9 ver.1 and 1.25 μg pSIA012 using Lipofectamine® 3000 (FIG. 12C).

The reporter cell line having a GUCY2D gene comprising a R838C mutation(Cas9 target site) fused to a blue fluorescent protein at thebeta-tubulin gene locus was transfected with 1.25 μg SIN-AAV SpCas9 ver.1 and 1.25 μg pSIA012 using Lipofectamine® 3000 (FIG. 12E).

FIG. 12B shows results obtained when the reporter cell line having awild-type GUCY2D gene (Cas9 target site) fused to a blue fluorescentprotein at the beta-tubulin gene locus was transfected with 1.25 μgSIN-AAV SpCas9 ver. 1 (FIG. 11A) and 1.25 μg pSIA015 (FIG. 11D) usingLipofectamine® 3000. pSIA015 is a plasmid comprising an AAV sequence(SEQ ID NO: 5507) that encodes for R838CH_Sp_T1 sgRNA (a sgRNAcomprising SEQ ID NO: 5398) and EGFP. SIN-AAV SpCas9 ver. 1 encodesSpCas9 and includes SIN sites (also called R838 target sites) located 5′(SEQ ID NO: 5478) and 3′ (SEQ ID NO: 5480) of the SpCas9 ORF. The 5′ SINsite (SEQ ID NO: 5478) in SIN-AAV SpCas9 ver. 1 comprises SEQ ID NO:5326, which is targeted by sgRNA comprising SEQ ID NO: 5398. The 3′ SINsite (SEQ ID NO: 5480) in SIN-AAV SpCas9 ver. 1 comprises SEQ ID NO:5368, which is targeted by sgRNA comprising SEQ ID NO: 5398 (Table 10).

TABLE 10 SIN-AAV SpCas9 ver. 1 comprising two SIN sitestargeted by a sgRNA comprising SEQ ID NO: 5398 SEQ ID Sequence Type NO:Sequences 5′ SIN site sequence 5478 ggaggatctgatccgggagcacacggaggagctgga Target sequence 5326 tctgatccgggagcacacgg3′ SIN site sequence 5480 tccagctcctccgtgtgctc ccggatcagatcctccTarget sequence 5368 ccgtgtgctcccggatcaga

The reporter cell line having a GUCY2D gene comprising a R838H mutation(Cas9 target site) fused to a blue fluorescent protein at thebeta-tubulin gene locus was transfected with 1.25 μg SIN-AAV SpCas9 ver.1 and 1.25 μg pSIA015 using Lipofectamine® 3000 (FIG. 12D).

The reporter cell line having a GUCY2D gene comprising a R838C mutation(Cas9 target site) fused to a blue fluorescent protein at thebeta-tubulin gene locus was transfected with 1.25 μg SIN-AAV SpCas9 ver.1 and 1.25 μg pSIA015 using Lipofectamine® 3000 (FIG. 12F).

FIG. 12G shows results obtained when the reporter cell line having awild-type GUCY2D gene (Cas9 target site) fused to a blue fluorescentprotein at the beta-tubulin gene locus was transfected with 1.25 μgSIN-AAV SpCas9 ver. 2 (FIG. 11B) and 1.25 μg pSIA012 (FIG. 11D) usingLipofectamine® 3000. SIN-AAV SpCas9 ver. 2 encodes SpCas9 and includesSIN sites located 5′ (SEQ ID NO: 5479) and 3′ (SEQ ID NO: 5480) of theSpCas9 ORF. The 5′ SIN site (SEQ ID NO: 5479) in SIN-AAV SpCas9 ver. 2comprises SEQ ID NO: 5327, which is targeted by sgRNA comprising SEQ IDNO: 5285. The 3′ SIN site (SEQ ID NO: 5480) in SIN-AAV SpCas9 ver. 2comprises SEQ ID NO: 5369, which is targeted by sgRNA comprising SEQ IDNO: 5285 (Table 11).

TABLE 11 SIN-AAV SpCas9 ver. 2 comprising two SIN sitestargeted by a sgRNA comprising SEQ ID NO: 5285 SEQ ID Sequence Type NO:Sequences 5′ SIN site sequence 5479 aggatctgatccgggagcac acggaggagctggaTarget sequence 5327 ggatctgatccgggagcaca 3′ SIN site sequence 5480tccagctcctccgtgtgctc ccggatcagatcctcc Target sequence 5369tgtgctcccggatcagatcc

The reporter cell line having a GUCY2D gene comprising a R838H mutation(Cas9 target site) fused to a blue fluorescent protein at thebeta-tubulin gene locus was transfected with 1.25 μg SIN-AAV SpCas9 ver.2 and 1.25 μg pSIA012 using Lipofectamine® 3000 (FIG. 12I).

The reporter cell line having a GUCY2D gene comprising a R838C mutation(Cas9 target site) fused to a blue fluorescent protein at thebeta-tubulin gene locus was transfected with 1.25 μg SIN-AAV SpCas9 ver.2 and 1.25 μg pSIA012 using Lipofectamine® 3000 (FIG. 12K).

FIG. 12H shows results obtained when the reporter cell line having awild-type GUCY2D gene (Cas9 target site) fused to a blue fluorescentprotein at the beta-tubulin gene locus was transfected with 1.25 μgSIN-AAV SpCas9 ver. 2 (FIG. 11B) and 1.25 μg pSIA015 (FIG. 11D) usingLipofectamine® 3000. SIN-AAV SpCas9 ver. 2 encodes SpCas9 and includesSIN sites located 5′ (SEQ ID NO: 5479) and 3′ (SEQ ID NO: 5480) of theSpCas9 ORF. The 5′ SIN site (SEQ ID NO: 5479) in SIN-AAV SpCas9 ver. 2comprises SEQ ID NO: 5326, which is targeted by sgRNA comprising SEQ IDNO: 5398. The 3′ SIN site (SEQ ID NO: 5480) in SIN-AAV SpCas9 ver. 2comprises SEQ ID NO: 5368, which is targeted by sgRNA comprising SEQ IDNO: 5398 (Table 12).

TABLE 12 SIN-AAV SpCas9 ver. 2 comprising two SIN sitestargeted by a sgRNA comprising SEQ ID NO: 5398 SEQ ID Sequence Type NO:Sequences 5′ SIN site sequence 5479 aggatctgatccgggagcac acggaggagctggaTarget sequence 5326 tctgatccgggagcacacgg 3′ SIN site sequence 5480tccagctcctccgtgtgctc ccggatcagatcctcc Target sequence 5368ccgtgtgctcccggatcaga

The reporter cell line having a GUCY2D gene comprising a R838H mutation(Cas9 target site) fused to a blue fluorescent protein at thebeta-tubulin gene locus was transfected with 1.25 μg SIN-AAV SpCas9 ver.2 and 1.25 μg pSIA015 using Lipofectamine® 3000 (FIG. 12J).

The reporter cell line having a GUCY2D gene comprising a R838C mutation(Cas9 target site) fused to a blue fluorescent protein at thebeta-tubulin gene locus was transfected with 1.25 μg SIN-AAV SpCas9 ver.2 and 1.25 μg pSIA015 using Lipofectamine® 3000 (FIG. 12L).

FIG. 12M shows results obtained when the reporter cell line having awild-type GUCY2D gene (Cas9 target site) fused to a blue fluorescentprotein at the beta-tubulin gene locus was transfected with 1.25 μgNon-SIN-AAV SpCas9 (FIG. 11C) and 1.25 μg pSIA012 (FIG. 11D) usingLipofectamine® 3000. Non-SIN-AAV SpCas9 encodes SpCas9 and includes noSIN sites (also called R838 target sites).

The reporter cell line having a GUCY2D gene comprising a R838H mutation(Cas9 target site) fused to a blue fluorescent protein at thebeta-tubulin gene locus was transfected with 1.25 μg Non-SIN-AAV SpCas9and 1.25 μg pSIA012 using Lipofectamine® 3000 (FIG. 12P).

The reporter cell line having a GUCY2D gene comprising a R838C mutation(Cas9 target site) fused to a blue fluorescent protein at thebeta-tubulin gene locus was transfected with 1.25 μg Non-SIN-AAV SpCas9and 1.25 μg pSIA012 using Lipofectamine® 3000 (FIG. 12S).

FIG. 12N shows results obtained when the reporter cell line having awild-type GUCY2D gene (Cas9 target site) fused to a blue fluorescentprotein at the beta-tubulin gene locus was transfected with 1.25 μgNon-SIN-AAV SpCas9 (FIG. 11C) and 1.25 μg pSIA015 (FIG. 11D) usingLipofectamine® 3000.

The reporter cell line having a GUCY2D gene comprising a R838H mutation(Cas9 target site) fused to a blue fluorescent protein at thebeta-tubulin gene locus was transfected with 1.25 μg Non-SIN-AAV SpCas9and 1.25 μg pSIA015 using Lipofectamine® 3000 (FIG. 12Q).

The reporter cell line having a GUCY2D gene comprising a R838C mutation(Cas9 target site) fused to a blue fluorescent protein at thebeta-tubulin gene locus was transfected with 1.25 μg Non-SIN-AAV SpCas9and 1.25 μg pSIA015 using Lipofectamine® 3000 (FIG. 12T).

The reporter cell line having a wild-type GUCY2D gene (Cas9 target site)fused to a blue fluorescent protein at the beta-tubulin gene locus wastransfected with only transfection reagent (no DNA) using Lipofectamine®3000 (FIG. 12O).

The reporter cell line having a GUCY2D gene comprising a R838H mutation(Cas9 target site) fused to a blue fluorescent protein at thebeta-tubulin gene locus was transfected with only transfection reagent(no DNA) using Lipofectamine® 3000 (FIG. 12R).

The reporter cell line having a GUCY2D gene comprising a R838C mutation(Cas9 target site) fused to a blue fluorescent protein at thebeta-tubulin gene locus was transfected with only transfection reagent(no DNA) using Lipofectamine® 3000 (FIG. 12U).

At 72 hours post-transfection, reporter cells were dissociated from theplates by incubation with trypsin-EDTA, and analyzed for bluefluorescence (BFP) and green fluorescence (GFP) by flow cytometry. Aframe-shift induced by genome editing at a Cas9 target site (whether theCas9 target site is the wild-type GUCY2D gene, the GUCY2D genecomprising the R838H mutation, or the GUCY2D gene comprising the R838Cmutation) of the HEK 293FT cell results in loss of BFP. EGFP and sgRNAare encoded on the same vector and the EGFP serves as a transfectionmarker. Therefore, HEK 293FT cells transfected with pSIA012 (whichcomprises an AAV sequence that encodes R838H_Sp_T2 sgRNA—a sgRNAcomprising SEQ ID NO: 5285) or pSIA015 (which comprises an AAV sequencethat encodes R838CH_Sp_T1 sgRNA—a sgRNA comprising SEQ ID NO: 5398) areGFP positive.

Gene editing in the transfected cells was estimated in the cellpopulations plotted within a gate (FIGS. 12A-12U). The boldedpercentages in FIGS. 12A-12U indicate BFP negative and GFP positivecells within the gate. BFP negative means that gene editing occurred atthe Cas9 target site of these transfected HEK 293FT cells. GFP positivemeans that these transfected HEK 293FT cells contain a plasmid thatencodes EGFP and sgRNA.

FIG. 12A shows that of the transfected HEK 293FT reporter cells thathave the wild-type GUCY2D gene as the Cas9 target site in the gate,9.63% of these cells had the wild-type GUCY2D gene edited whentransfected with SIN-AAV SpCas9 ver. 1 and pSIA012, which encodesR838H_Sp_T2 sgRNA (sgRNA comprising SEQ ID NO: 5285).

FIG. 12B shows that of the transfected HEK 293FT reporter cells thathave the wild-type GUCY2D gene as the Cas9 target site in the gate, ˜0%of these cells had the wild-type GUCY2D gene edited when transfectedwith SIN-AAV SpCas9 ver. 1 and pSIA015, which encodes R838CH_Sp_T1 sgRNA(sgRNA comprising SEQ ID NO: 5398).

FIG. 12C shows that of the transfected HEK 293FT reporter cells thathave the R838H mutation as the Cas9 target site in the gate, 53.07% ofthese cells had the R838H mutation edited when transfected with SIN-AAVSpCas9 ver. 1 and pSIA012.

FIG. 12D shows that of the transfected HEK 293FT reporter cells thathave the R838H mutation as the Cas9 target site in the gate, 21.04% ofthese cells had the R838H mutation edited when transfected with SIN-AAVSpCas9 ver. 1 and pSIA015.

FIG. 12E shows that of the transfected HEK 293FT reporter cells thathave the R838C mutation as the Cas9 target site in the gate, ˜0% ofthese cells had the R838C mutation edited when transfected with SIN-AAVSpCas9 ver. 1 and pSIA012.

FIG. 12F shows that of the transfected HEK 293FT reporter cells thathave the R838C mutation as the Cas9 target site in the gate, 26.35% ofthese cells had the R838C mutation edited when transfected with SIN-AAVSpCas9 ver. 1 and pSIA015.

FIG. 12G shows that of the transfected HEK 293FT reporter cells thathave the wild-type GUCY2D gene as the Cas9 target site in the gate,7.43% of these cells had the wild-type GUCY2D gene edited whentransfected with SIN-AAV SpCas9 ver. 2 and pSIA012.

FIG. 12H shows that of the transfected HEK 293FT reporter cells thathave the wild-type GUCY2D gene as the Cas9 target site in the gate, ˜0%of these cells had the wild-type GUCY2D gene edited when transfectedwith SIN-AAV SpCas9 ver. 2 and pSIA015.

FIG. 12I shows that of the transfected HEK 293FT reporter cells thathave the R838H mutation as the Cas9 target site in the gate, 52.62% ofthese cells had the R838H mutation edited when transfected with SIN-AAVSpCas9 ver. 2 and pSIA012.

FIG. 12J shows that of the transfected HEK 293FT reporter cells thathave the R838H mutation as the Cas9 target site in the gate, 18.50% ofthese cells had the R838H mutation edited when transfected with SIN-AAVSpCas9 ver. 2 and pSIA015.

FIG. 12K shows that of the transfected HEK 293FT reporter cells thathave the R838C mutation as the Cas9 target site in the gate, ˜0% ofthese cells had the R838C mutation edited when transfected with SIN-AAVSpCas9 ver. 2 and pSIA012.

FIG. 12L shows that of the transfected HEK 293FT reporter cells thathave the R838C mutation as the Cas9 target site in the gate, 24.73% ofthese cells had the R838C mutation edited when transfected with SIN-AAVSpCas9 ver. 2 and pSIA015.

FIG. 12M shows that of the transfected HEK 293FT reporter cells thathave the wild-type GUCY2D gene as the Cas9 target site in the gate,13.51% of these cells had the wild-type GUCY2D gene edited whentransfected with Non-SIN-AAV SpCas9 and pSIA012.

FIG. 12N shows that of the transfected HEK 293FT reporter cells thathave the wild-type GUCY2D gene as the Cas9 target site in the gate, ˜0%of these cells had the wild-type GUCY2D gene edited when transfectedwith Non-SIN-AAV SpCas9 and pSIA015.

FIG. 12O shows that of the transfected HEK 293FT reporter cells thathave the wild-type GUCY2D gene as the Cas9 target site in the gate, 0%of these cells had the wild-type GUCY2D gene edited when no DNA wasused.

FIG. 12P shows that of the transfected HEK 293FT reporter cells thathave the R838H mutation as the Cas9 target site in the gate, 50.34% ofthese cells had the R838H mutation edited when transfected withNon-SIN-AAV SpCas9 and pSIA012.

FIG. 12Q shows that of the transfected HEK 293FT reporter cells thathave the R838H mutation as the Cas9 target site in the gate, 20.62% ofthese cells had the R838H mutation edited when transfected withNon-SIN-AAV SpCas9 and pSIA015.

FIG. 12R shows that of the transfected HEK 293FT reporter cells thathave the R838H mutation as the Cas9 target site in the gate, 0% of thesecells had the R838H mutation edited when no DNA was used.

FIG. 12S shows that of the transfected HEK 293FT reporter cells thathave the R838C mutation as the Cas9 target site in the gate, ˜0% ofthese cells had the R838C mutation edited when transfected withNon-SIN-AAV SpCas9 and pSIA012.

FIG. 12T shows that of the transfected HEK 293FT reporter cells thathave the R838C mutation as the Cas9 target site in the gate, 29.17% ofthese cells had the R838C mutation edited when transfected withNon-SIN-AAV SpCas9 and pSIA015.

FIG. 12U shows that of the transfected HEK 293FT reporter cells thathave the R838C mutation as the Cas9 target site in the gate, 0% of thesecells had the R838C mutation edited when no DNA was used.

Since no genome editing was induced in mock transfected cells, the vastmajority of mock transfected cells are BFP positive (FIGS. 12O, 12R, and12U).

The results reported in FIGS. 12A-U provide evidence that the SINvectors of the present disclosure can edit genomic target alleles withspecificity.

To determine the ability of SIN vectors to limit Cas9 expression, theexpression levels of Cas9 protein were measured by immunoblot (FIGS.13A-C) for the cells used in the experiments described by FIGS. 12A-U.

At 72 hours post-transfection, reporter cells were also harvested inPBS, and total protein was extracted in 0.1% Triton X-100/TBS (25 mMTris-HCl (pH 7.5) and 150 mM NaCl). Five micrograms of total protein wasseparated on NUPAGE 4-12% polyacrylamide/Tris-Bis gels, and transferredonto nitrocellulose membranes. SpCas9, EGFP (as a transfection control)and beta actin (as an internal control) were detected using a Cas9monoclonal antibody, GFP Tag polyclonal antibody, and beta actin loadingcontrol monoclonal antibody, respectively.

FIG. 13A (lane 1) shows Cas9 inactivation in HEK 293FT reporter cellsthat have the wild-type GUCY2D gene as the Cas9 target site and thatwere transfected with SIN-AAV SpCas9 ver. 1 (FIG. 11A) and pSIA012,which encodes R838H_Sp_T2 sgRNA (sgRNA comprising SEQ ID NO: 5285).

FIG. 13A (lane 2) shows Cas9 inactivation in HEK 293FT reporter cellsthat have the wild-type GUCY2D gene as the Cas9 target site and thatwere transfected with SIN-AAV SpCas9 ver. 2 (FIG. 11B) and pSIA012.

FIG. 13A (lane 3) shows Cas9 expression in HEK 293FT reporter cells thathave the wild-type GUCY2D gene as the Cas9 target site and that weretransfected with Non-SIN-AAV SpCas9 (FIG. 11C) and pSIA012.

FIG. 13A (lane 4) shows no Cas9 expression in HEK 293FT reporter cellsthat have the wild-type GUCY2D gene as the Cas9 target site and thatwere not transfected with any DNA.

FIG. 13A (lane 5) shows Cas9 inactivation in HEK 293FT reporter cellsthat have the wild-type GUCY2D gene as the Cas9 target site and thatwere transfected with SIN-AAV SpCas9 ver. 1 (FIG. 11A) and pSIA015,which encodes R838CH_Sp_T1 sgRNA (sgRNA comprising SEQ ID NO: 5398).

FIG. 13A (lane 6) shows Cas9 inactivation in HEK 293FT reporter cellsthat have the wild-type GUCY2D gene as the Cas9 target site and thatwere transfected with SIN-AAV SpCas9 ver. 2 (FIG. 11B) and pSIA015.

FIG. 13A (lane 7) shows Cas9 expression in HEK 293FT reporter cells thathave the wild-type GUCY2D gene as the Cas9 target site and that weretransfected with Non-SIN-AAV SpCas9 (FIG. 11C) and pSIA015.

FIG. 13B (lane 1) shows Cas9 inactivation in HEK 293FT reporter cellsthat have the R838H mutation as the Cas9 target site and that weretransfected with SIN-AAV SpCas9 ver. 1 (FIG. 11A) and pSIA012, whichencodes R838H_Sp_T2 sgRNA (sgRNA comprising SEQ ID NO: 5285).

FIG. 13B (lane 2) shows Cas9 inactivation in HEK 293FT reporter cellsthat have the R838H mutation as the Cas9 target site and that weretransfected with SIN-AAV SpCas9 ver. 2 (FIG. 11B) and pSIA012.

FIG. 13B (lane 3) shows Cas9 expression in HEK 293FT reporter cells thathave the R838H mutation as the Cas9 target site and that weretransfected with Non-SIN-AAV SpCas9 (FIG. 11C) and pSIA012.

FIG. 13B (lane 4) shows no Cas9 expression in HEK 293FT reporter cellsthat have the R838H mutation as the Cas9 target site and that were nottransfected with any DNA.

FIG. 13B (lane 5) shows Cas9 inactivation in HEK 293FT reporter cellsthat have the R838H mutation as the Cas9 target site and that weretransfected with SIN-AAV SpCas9 ver. 1 (FIG. 11A) and pSIA015, whichencodes R838CH_Sp_T1 sgRNA (sgRNA comprising SEQ ID NO: 5398).

FIG. 13B (lane 6) shows Cas9 inactivation in HEK 293FT reporter cellsthat have the R838H mutation as the Cas9 target site and that weretransfected with SIN-AAV SpCas9 ver. 2 (FIG. 11B) and pSIA015.

FIG. 13B (lane 7) shows Cas9 expression in HEK 293FT reporter cells thathave the R838H mutation as the Cas9 target site and that weretransfected with Non-SIN-AAV SpCas9 (FIG. 11C) and pSIA015.

FIG. 13C (lane 1) shows Cas9 inactivation in HEK 293FT reporter cellsthat have the R838C mutation as the Cas9 target site and that weretransfected with SIN-AAV SpCas9 ver. 1 (FIG. 11A) and pSIA012, whichencodes R838H_Sp_T2 sgRNA (sgRNA comprising SEQ ID NO: 5285).

FIG. 13C (lane 2) shows Cas9 inactivation in HEK 293FT reporter cellsthat have the R838C mutation as the Cas9 target site and that weretransfected with SIN-AAV SpCas9 ver. 2 (FIG. 11B) and pSIA012.

FIG. 13C (lane 3) shows Cas9 expression in HEK 293FT reporter cells thathave the R838C mutation as the Cas9 target site and that weretransfected with Non-SIN-AAV SpCas9 (FIG. 11C) and pSIA012.

FIG. 13C (lane 4) shows no Cas9 expression in HEK 293FT reporter cellsthat have the R838C mutation as the Cas9 target site and that were nottransfected with any DNA.

FIG. 13C (lane 5) shows Cas9 inactivation in HEK 293FT reporter cellsthat have the R838C mutation as the Cas9 target site and that weretransfected with SIN-AAV SpCas9 ver. 1 (FIG. 11A) and pSIA015, whichencodes R838CH_Sp_T1 sgRNA (sgRNA comprising SEQ ID NO: 5398).

FIG. 13C (lane 6) shows Cas9 inactivation in HEK 293FT reporter cellsthat have the R838C mutation as the Cas9 target site and that weretransfected with SIN-AAV SpCas9 ver. 2 (FIG. 11B) and pSIA015.

FIG. 13C (lane 7) shows Cas9 expression in HEK 293FT reporter cells thathave the R838C mutation as the Cas9 target site and that weretransfected with Non-SIN-AAV SpCas9 (FIG. 11C) and pSIA015.

To confirm that introduced SIN sites do not influence transcription andtranslation of Cas9, HEK 293FT cells were transfected with 1.25 μg ofpDL107 (which encodes GFP and does not encode sgRNA) and either (1)SIN-AAV SpCas9 ver. 1, (2) SIN-AAV SpCas9 ver. 2, or (3) Non-SIN-AAVSpCas9. Cells were seeded in 2.5 ml of 10% FBS/DMEM at 500,000 cells perwell in 6-well plates at 24 hours before transfection. At 72 hours aftertransfection, GFP expression of all the transfected cells were analyzedby flow cytometry, and total protein was extracted in 0.1% TritonX-100/TBS (25 mM Tris-HCl (pH 7.5) and 150 mM NaCl). Five micrograms oftotal protein was separated on a NUPAGE 4-12% polyacrylamide/Tris-Bisgel, and transferred onto nitrocellulose membranes. SpCas9, EGFP (as atransfection control) and beta actin (as an internal control) weredetected using a Cas9 monoclonal antibody, GFP Tag polyclonal antibodyand beta actin loading control monoclonal antibody, respectively.Results showed that there was equal SpCas9 expression in HEK 293FT cellstransfected with (1) SIN-AAV SpCas9 ver. 1, (2) SIN-AAV SpCas9 ver. 2,and (3) Non-SIN-AAV SpCas9 (Data not shown).

SIN Cas9 vectors showed decreased expression of Cas9 in all threereproter cell lines when targeted by either a guide RNA comprising SEQID NO: 5285 or 5398, thus providing evidence that SIN vectors of thepresent disclosure can limit expression of Cas9 protein (FIGS. 13A-C),while still causing editing of targeted alleles (FIGS. 12A-U).

Example 31—Self-Inactivating (SIN) CRISPR-Cas Systems

To determine the ability of SIN vectors to limit Cas9 expression invivo, the expression levels of Cas9 protein were measured by immunoblotin mouse retinas isolated 28 days after subretinal AAV injection (FIGS.15A-B).

Eight to ten-week-old C57BL/6J mice were purchased from JacksonLaboratories and maintained at MisoPro Animal Facility (Cambridge,Mass.). All animal procedures were conducted in compliance with theAnimal Welfare Act, and the Guide for the Care and Use of LaboratoryAnimals, the Office of Laboratory Animal Welfare and in accordance withthe Association for Research in Vision and Ophthalmology (ARVO)Statement for the Use of Animals in Ophthalmic and Vision Research.

AAVs were delivered by subretinal injection [2×10⁹ genome copies (GC)for each vector] into the wild-type mice following standard subretinalinjection procedure. There were 10 different AAV groups delivered bysubretinal injection. The first group included: SIN-AAV SpCas9 ver. 1(FIG. 14A), AAV-R838H_Sp_T2 sgRNA, and an AAV comprising a R838Hmutation within the GUCY2D gene. FIG. 11D shows the structuralarrangement of an AAV sequence located within pSIA012, a plasmid that isused to generate AAV-R838H_Sp_T2 sgRNA. AAV-R838H_Sp_T2 sgRNA encodes asgRNA comprising SEQ ID NO: 5285. The second group included: SIN-AAVSpCas9 ver. 2 (FIG. 14B), AAV-R838H_Sp_T2 sgRNA, and an AAV comprising aR838H mutation within the GUCY2D gene. The third group included:Non-SIN-AAV SpCas9 (FIG. 14C), AAV-R838H_Sp_T2 sgRNA, and an AAVcomprising a R838H mutation within the GUCY2D gene. The fourth groupincluded: AAV-R838H_Sp_T2 sgRNA and an AAV comprising a R838H mutationwithin the GUCY2D gene. The fifth group included: AAV-R838H_Sp_T2 sgRNA.The sixth group included: SIN-AAV SpCas9 ver. 1 (FIG. 14A), AAVR838CH_Sp_T1 sgRNA, and an AAV comprising a R838H mutation within theGUCY2D gene. FIG. 11D shows the structural arrangement of an AAVsequence located within pSIA015, a plasmid that is used to generateAAV-R838CH_Sp_T1 sgRNA. AAV-R838CH_Sp_T1 sgRNA encodes a sgRNAcomprising SEQ ID NO: 5398. The seventh group included: SIN-AAV SpCas9ver. 2 (FIG. 14B), AAV R838CH_Sp_T1 sgRNA, and an AAV comprising a R838Hmutation within the GUCY2D gene. The eighth group included: Non-SIN-AAVSpCas9 (FIG. 14C), AAV R838CH_Sp_T1 sgRNA, and an AAV comprising a R838Hmutation within the GUCY2D gene. The ninth group included: AAVR838CH_Sp_T1 sgRNA and an AAV comprising a R838H mutation within theGUCY2D gene. The tenth group included: AAV R838CH_Sp_T1 sgRNA.

On Day 28 post AAV injection, mouse retinas were isolated usingmicro-dissecting scissors under a dissection microscope. The retinalpigment epithelium (RPE) layer was carefully removed. Cells were lysedin RIPA lysis buffer (Thermo Fisher Scientific) supplemented with HaltProtease Inhibitor cocktail on ice. Protein quantification was measuredby Pierce BCA assay (Thermo Fisher Scientific).

Protein per retina sample was separated using the NuPAGE ElectrophoresisSystem (Thermo Fisher Scientific), after which the proteins weretransferred using 0.45 um Pore Size Nitrocellulose Membrane Filter PaperSandwich (Thermo Fisher Scientific). Blocking buffer with Pierce TBST(Tris-buffered saline with Tween 20 detergent) buffer containing 5%(w/v) BSA was prepared. Membranes were blocked by Pierce TBST(Tris-buffered saline with Tween 20 detergent) buffer containing 5%(w/v) BSA at room temperature for 2 hours, or 4° C. overnight. Themembranes were then incubated with a primary antibody by diluting theantibody with blocking buffer and incubating the membrane on a rocker at4° C. overnight. After three washes, the membrane was incubated withsecondary antibodies at 1:5000 at RT for 1 hour. Imaging was obtainedwith ChemiDoc™ MP Imaging System (BioRad).

FIG. 15A (lanes 1-2) shows Cas9 inactivation in retinas (without RPE)that were transfected with SIN-AAV SpCas9 ver. 1, AAV R838H_Sp_T2 sgRNA,and an AAV comprising a R838H mutation within the GUCY2D gene.

FIG. 15A (lanes 3-4) shows Cas9 inactivation in retinas (without RPE)that were transfected with SIN-AAV SpCas9 ver. 2, AAV R838H_Sp_T2 sgRNA,and an AAV comprising a R838H mutation within the GUCY2D gene.

FIG. 15A (lanes 5-6) shows Cas9 expression in retinas (without RPE) thatwere transfected with Non-SIN-AAV SpCas9, AAV R838H_Sp_T2 sgRNA, and anAAV comprising a R838H mutation within the GUCY2D gene.

FIG. 15A (lanes 7-8) shows no Cas9 expression in retinas (without RPE)that were transfected with AAV R838H_Sp_T2 sgRNA, and an AAV comprisinga R838H mutation within the GUCY2D gene.

FIG. 15A (lanes 9-10) shows no Cas9 expression in retinas (without RPE)that were transfected with only AAV R838H_Sp_T2 sgRNA.

FIG. 15B (lanes 1-2) shows Cas9 inactivation in retinas (without RPE)that were transfected with SIN-AAV SpCas9 ver. 1, AAV R838CH_Sp_T1sgRNA, and an AAV comprising a R838H mutation within the GUCY2D gene.

FIG. 15B (lanes 3-4) shows Cas9 inactivation in retinas (without RPE)that were transfected with SIN-AAV SpCas9 ver. 2, AAV R838CH_Sp_T1sgRNA, and an AAV comprising a R838H mutation within the GUCY2D gene.

FIG. 15B (lanes 5-6) show Cas9 expression in retinas (without RPE) thatwere transfected with Non-SIN-AAV SpCas9, AAV R838CH_Sp_T1 sgRNA, and anAAV comprising a R838H mutation within the GUCY2D gene. FIG. 15B (lane6) shows a fainter Cas9 band than in lane 5, but the Cas9 band is stillpresent. The retina in lane 6 was not transduced as well as the retinain lane 5. In fact the band for GFP is also fainter in lane 6 comparedto lane 5 further demonstrating that the retina in lane 6 was nottransduced as well as the retina in lane 5.

FIG. 15B (lanes 7-8) shows no Cas9 expression in retinas (without RPE)that were transfected with AAV R838CH_Sp_T1 sgRNA and an AAV comprisinga R838H mutation within the GUCY2D gene.

FIG. 15B (lanes 9-10) shows no Cas9 expression in retinas (without RPE)that were transfected with only AAV R838CH_Sp_T1 sgRNA.

As discussed above, two versions of self-inactivating (SIN) AAV vectorsthat limit their own expression of Cas9 after transfection were created.Examples of version 1 vectors are depicted in FIGS. 11A and 14A.Examples of version 2 vectors are depicted in FIGS. 11B and 14B. Bothversion 1 and version 2 vectors comprise two SIN sites (also called R838target sites), which are vulnerable to cutting by Cas9-sgRNA RNPs.Cas9-mediated double strand breaks at one of these sites could lead toremoval of either a promoter or polyadenylation signal in the Cas9 gene.Cas9-mediated double strand breaks at both of these sites occurringcontemporaneously could lead to deletion of the Cas9 gene. Bothpossibilities (e.g., 1 or 2 DSBs) would inhibit Cas9 expression.

It has been observed that version 1 vectors lead to more efficientself-inactivation than version 2 vectors (FIG. 15B). Version 1 vectorscomprise a 5′ SIN site (R838 target site) that is located upstream ofthe Cas9 open reading frame (ORF) and downstream of a SV40 nuclearlocalization signal (NLS). Version 2 vectors comprise a 5′ SIN site(R838 target site) that is located upstream of the Cas9 open readingframe (ORF) and upstream of a SV40 nuclear localization signal (NLS)within a 5′ untranslated region (UTR). In a version 1 vector, mutationsresulting from non-homologous end-joining could create frame-shifts,which cause introduction of premature stop codons in the Cas9 gene ORF.In a version 2 vector, such mutations would be unlikely to create suchchanges in the Cas9 ORF since the SIN site (R838 target site) is in the5′ UTR, outside of the ORF. These mutations could still disrupttranscription initiation, but their overall effect on Cas9 expression islikely to be less than the mutations in a version 1 vector. Furthermore,once a mutation is created in either vector, a second is unlikely sincethe site will no longer share sufficient homology with the sgRNA spacersequence for efficient additional editing. For at least these reasons,there is a disparity in SIN efficiency observed between the two vectorversions.

SIN Cas9 vectors showed decreased expression of Cas9 when targeted byeither a guide RNA comprising SEQ ID NO: 5285 or 5398, thus providingevidence that SIN vectors of the present disclosure can limit expressionof Cas9 protein in vivo.

Example 32—Testing of Guide RNAs in Cells for On-Target Activity

To further evaluate the specificity of gRNAs provided herein, selectedgRNAs were further tested for on-target activity in immortalized humanpatient-derived fibroblasts that have a R838H mutant allele as a copy ofthe GUCY2D gene.

Patients with a R838H mutant allele provided skin biopsies to create animmortalized cell line. Primary fibroblasts were isolated from thesebiopsies and cultured in 10% FBS/DMEM supplemented with GlutaMAX™, asupplement comprising an L-alanyl-L-glutamine dipeptide in 0.85% NaClmanufactured by Thermo Fisher Scientific, Massachusetts, US. GlutaMAX™is. Immortalization of patient-derived fibroblasts was conducted by theNatural and Medical Sciences Institute at the University of Tubingen,Germany. The immortalized patient-derived fibroblasts were cultured in10% FBS/DMEM supplemented with GlutaMAX™ and passaged every 3-4 days.

A plasmid, pSpCas9 (BB)-2A-miRFP670 (“pSpCas9”) (SEQ ID NO: 5512), wasobtained (Addgene: Watertown, Mass.). The pSpCas9 sequence contains aCMV promoter-driven SpCas9 gene, SV40 polyadenylation signal, 2A linkerpeptide, and miRFP670. The 2A linker peptide is located on the plasmidbetween the SpCas9 gene and miRFP670 and is cleaved after translation.Therefore, if the SpCas9 gene is transcribed and translated, then RFP isalso transcribed and translated.

pSIA012 (SEQ ID NO: 5469), depicted in FIG. 11D, comprises a sequencethat encodes for a U6 promoter driven R838H_Sp_T2 sgRNA (a sgRNAcomprising SEQ ID NO: 5285) and CMV promoter driven EGFP.

pSIA015 (SEQ ID NO: 5471), depicted in FIG. 11D, comprises a sequencethat encodes for a U6 promoter driven R838CH_Sp_T1 sgRNA (a sgRNAcomprising SEQ ID NO: 5398) and CMV promoter driven EGFP.

As described in Example 24, pSIA003 is a plasmid that can encode a U6driven gRNA sequence depending on the gRNA sequence that is cloned intothe plasmid. For this particular experiment, the pSIA003 that was usedcomprises a sequence that encodes for a U6 promoter driven non-targeting(e.g., scrambled) sgRNA that does not target the R838H mutant allele.The non-targeting sgRNA comprises SEQ ID NO: 5513. pSIA003 alsocomprises a sequence that encodes a CMV promoter driven EGFP.

Immortalized patient-derived fibroblasts comprising a R838H mutantallele were seeded in 2.5 ml of 10% FBS/DMEM supplemented with GlutaMAX™at 500,000 cells per well in 6-well plates 24 hours before transfectionvia electroporation.

The immortalized patient-derived fibroblast cells were transfected with10 μg of pSpCas9 and 1 μg of either: pSIA012, pSIA015, or pSIA003, usinga NEON™ electroporation system (available from Thermo Fisher Scientific,Massachusetts, US).

At 48 hours post-transfection, immortalized patient-derived fibroblastcells were dissociated from the plates by incubation with trypsin-EDTA,and analyzed for red fluorescence (RFP) and green fluorescence (GFP) byflow cytometry. Each of the three plasmids that encodes the sgRNAs usedin this Example encodes EGFP, and the EGFP serves as a transfectionmarker. Immortalized patient-derived fibroblasts transfected withpSIA012, pSIA015, or pSIA003 are GFP positive. RFP and SpCas9 areencoded on the same plasmid and the RFP serves as a transfection marker.Immortalized patient-derived fibroblasts transfected with pSpCas9 areRFP positive.

Immortalized patient-derived fibroblasts that are GFP+RFP+ are cellsthat were successfully transfected with one of the three sgRNA plasmids(pSIA012, pSIA015, or pSIA003) and also with pSpCas9. Genomic DNA wasextracted from sorted GFP+RFP+ cells. Indels were analyzed by TIDE todetermine editing efficiency in the immortalized patient-derivedfibroblasts transfected with pSpCas9 and either pSIA012 (FIG. 16, sample1), pSIA015 (FIG. 16, sample 2), or pSIA003 (FIG. 16, sample 3).

FIG. 16, sample 1 shows that of the GFP+RFP+ transfected immortalizedpatient-derived fibroblasts that have the R838H mutant allele,33.9±10.8% of these cells had the R838H mutant allele edited whenR838H_Sp_T2 sgRNA (sgRNA comprising SEQ ID NO: 5285) was used as thesgRNA. The 33.9+10.8% editing efficiency was the result of threeseparate experiments that included immortalized patient derivedfibroblasts from two separate patients. Patient #1's fibroblasts wereused in two experiments and Patent #2's fibroblasts were used in oneexperiment. Two different patients showed consistent ˜40% editing inspite of variability that can be seen from one patient to the next. Thissuggests that a gRNA or sgRNA comprising SEQ ID NO: 5285 consistentlyedits the R838H mutant allele.

FIG. 16, sample 2 shows that of the GFP+RFP+ transfected immortalizedpatient-derived fibroblasts that have the R838H mutant allele, 15.5±8.9%of these cells had the R838H mutant allele edited when R838CH_Sp_T1sgRNA (sgRNA comprising SEQ ID NO: 5398) was used as the sgRNA.

FIG. 16, sample 3 shows that of the GFP+RFP+ transfected immortalizedpatient-derived fibroblasts that have the R838H mutant allele, 1.15±0.3%of these cells had the R838H mutant allele edited when a non-targeting(e.g., scrambled) sgRNA that does not target the R838H mutant allele wasused as the sgRNA. This sample served as a negative control.

These data (presented in FIG. 16) provide evidence that sgRNAs of thepresent disclosure can effectively edit the mutant R838H GUCY2D gene inhuman cells.

Example 33—Testing of Guide RNAs in Cells for On-Target Activity

To further evaluate the specificity of gRNAs provided herein, selectedgRNAs were further tested for on-target activity in immortalized humanpatient-derived fibroblasts that have a R838C mutant allele as a copy ofthe GUCY2D gene.

Patients with a R838C mutant allele provided skin biopsies to create animmortalized cell line. Primary fibroblasts were isolated from thesebiopsies and cultured in 10% FBS/DMEM supplemented with GlutaMAX™.Immortalization of patient-derived fibroblasts was conducted by theNatural and Medical Sciences Institute at the University of Tubingen,Germany. The immortalized patient-derived fibroblasts were cultured in10% FBS/DMEM supplemented with GlutaMAX™ and passaged every 3-4 days.

Immortalized patient-derived fibroblasts comprising a R838C mutantallele were seeded in 2.5 ml of 10% FBS/DMEM supplemented with GlutaMAX™at 500,000 cells per well in 6-well plates 24 hours before transfectionvia electroporation.

The immortalized patient-derived fibroblast cells were transfected with10 μg of pSpCas9 and 1 μg of either: pSIA012, pSIA015, or pSIA003, usinga NEON™ electroporation system (available from Thermo Fisher Scientific,Massachusetts, US).

At 48 hours post-transfection, immortalized patient-derived fibroblastcells were dissociated from the plates by incubation with trypsin-EDTA,and analyzed for red fluorescence (RFP) and green fluorescence (GFP) byflow cytometry. Each of the three plasmids that encodes the sgRNAs usedin this Example encodes EGFP, and the EGFP serves as a transfectionmarker. Immortalized patient-derived fibroblasts transfected withpSIA012, pSIA015, or pSIA003 are GFP positive. RFP and SpCas9 areencoded on the same plasmid and the RFP serves as a transfection marker.Immortalized patient-derived fibroblasts transfected with pSpCas9 areRFP positive.

Immortalized patient-derived fibroblasts that are GFP+RFP+ are cellsthat were successfully transfected with one of the three sgRNA plasmids(pSIA012, pSIA015, or pSIA003) and also with pSpCas9. Genomic DNA wasextracted from sorted GFP+RFP+ cells. Indels were analyzed by TIDE todetermine editing efficiency in the immortalized patient-derivedfibroblasts transfected with pSpCas9 and either pSIA012 (FIG. 17, sample1), pSIA015 (FIG. 17, sample 2), or pSIA003 (FIG. 17, sample 3).

FIG. 17, sample 1 shows that of the GFP+RFP+ transfected immortalizedpatient-derived fibroblasts that have the R838C mutant allele,10.98±2.6% of these cells had the R838C mutant allele edited whenR838H_Sp_T2 sgRNA (sgRNA comprising SEQ ID NO: 5285) was used as thesgRNA.

FIG. 17, sample 2 shows that of the GFP+RFP+ transfected immortalizedpatient-derived fibroblasts that have the R838C mutant allele,34.15±0.6% of these cells had the R838C mutant allele edited whenR838CH_Sp_T1 sgRNA (sgRNA comprising SEQ ID NO: 5398) was used as thesgRNA.

FIG. 17, sample 3 shows that of the GFP+RFP+ transfected immortalizedpatient-derived fibroblasts that have the R838C mutant allele, 8.75±6.9%of these cells had the R838C mutant allele edited when a non-targeting(e.g., scrambled) sgRNA that does not target the R838C mutant allele wasused as the sgRNA. This sample served as a negative control.

These data (presented in FIG. 17) provide evidence that sgRNAs of thepresent disclosure can effectively edit the mutant R838C GUCY2D gene inhuman cells.

Example 34—Testing of Guide RNAs in Cells for Off-Target Activity

To further evaluate the specificity of gRNAs provided herein, selectedgRNAs were further tested for off-target activity in BJ-5TA HDF cells,which are hTERT-immortalized human fibroblast cells that are homozygousfor the wild-type copy of the GUCY2D gene (“wild-type fibroblasts”).

Wild-type fibroblasts were obtained (as ATCC® CRL-4001TM from ATCC,Manassas, Va.) and were cultured in a 4:1 mixture of supplemented DMEMand Medium 199 and passaged every 3-4 days. DMEM was supplemented with 4mM L-glutamine, 4.5 g/L glucose, and 1.5 g/L sodium bicarbonate. Medium199 was supplemented with 0.01 mg/mL hygromycin B and 10% FBS.

pSpCas9, pSIA012, pSIA015, and pSIA003, previously described in Example32 were used in this experiment. pSIA022 was also used. pSIA022 (SEQ IDNO: 5514), comprises a sequence that encodes for a U6 promoter drivenWT_T1_sgRNA (a sgRNA comprising SEQ ID NO: 5274) and CMV promoter drivenEGFP.

Wild-type fibroblasts were seeded in 2.5 ml of the 4:1 mixture ofsupplemented DMEM and Medium 199 at 500,000 cells per well in 6-wellplates 24 hours before transfection via electroporation.

Wild-type fibroblasts were transfected with 10 μg of pSpCas9 and 1 μg ofeither: pSIA012, pSIA015, pSIA022, or pSIA003, using a NEON™electroporation system (available from Thermo Fisher Scientific,Massachusetts, US).

At 48 hours post-transfection, wild-type fibroblasts were dissociatedfrom the plates by incubation with trypsin-EDTA, and analyzed for redfluorescence (RFP) and green fluorescence (GFP) by flow cytometry. Eachof the four plasmids that encodes the sgRNAs used in this Exampleencodes EGFP, and the EGFP serves as a transfection marker. Wild-typefibroblasts transfected with pSIA012, pSIA015, pSIA022, or pSIA003 areGFP positive. RFP and SpCas9 are encoded on the same plasmid and the RFPserves as a transfection marker. Wild-type fibroblasts transfected withpSpCas9 are RFP positive.

Wild-type fibroblasts that are GFP+RFP+ are cells that were successfullytransfected with one of the four sgRNA plasmids (pSIA012, pSIA015,pSIA022, or pSIA003) and also with pSpCas9. Genomic DNA was extractedfrom sorted GFP+RFP+ cells. Indels were analyzed by TIDE to determineediting efficiency in the wild-type fibroblasts transfected with pSpCas9and either pSIA012 (FIG. 18, sample 1), pSIA015 (FIG. 18, sample 2),pSIA022 (FIG. 18, sample 3), or pSIA003 (FIG. 18, sample 4).

FIG. 18, sample 1 shows that of the GFP+RFP+ transfected wild-typefibroblasts, 9.3±6.0% of these cells had the wild-type GUCY2D alleleedited when R838H_Sp_T2 sgRNA (sgRNA comprising SEQ ID NO: 5285) wasused as the sgRNA.

FIG. 18, sample 2 shows that of the GFP+RFP+ transfected wild-typefibroblasts, 1.9±0.7% of these cells had the wild-type GUCY2D alleleallele edited when R838CH_Sp_T1 sgRNA (sgRNA comprising SEQ ID NO: 5398)was used as the sgRNA.

FIG. 18, sample 3 shows that of the GFP+RFP+ transfected wild-typefibroblasts, 79.7±12.7% of these cells had the wild-type GUCY2D alleleallele edited when WT_Sp_T1 sgRNA (sgRNA comprising SEQ ID NO: 5274) wasused as the sgRNA. This sample served as a positive control.

FIG. 18, sample 4 shows that of the GFP+RFP+ transfected wild-typefibroblasts, 1.8±0.3% of these cells had the wild-type GUCY2D alleleedited when a non-targeting (e.g., scrambled) sgRNA that does not targetthe wild-type GUCY2D allele was used as the sgRNA. This sample served asa negative control.

These data provide evidence that sgRNAs of the present disclosure canhave minimal off-target activity in human cells.

NOTE REGARDING ILLUSTRATIVE EXAMPLES

While the present disclosure provides descriptions of various specificaspects for the purpose of illustrating various examples of the presentdisclosure and/or its potential applications, it is understood thatvariations and modifications will occur to those skilled in the art.Accordingly, the invention or inventions described herein should beunderstood to be at least as broad as they are claimed, and not as morenarrowly defined by particular illustrative examples provided herein.

1. A method for editing a guanylate cyclase 2D (GUCY2D) gene in a humancell, the method comprising: introducing into the human cell one or moredeoxyribonucleic acid (DNA) endonucleases to effect one or moresingle-strand breaks (SSBs) or double-strand breaks (DSBs) within ornear the GUCY2D gene or other DNA sequences that encode regulatoryelements of the GUCY2D gene that results in a deletion, insertion, orcorrection thereby creating an edited human cell. 2.-4. (canceled) 5.The method of claim 1, wherein the one or more DNA endonucleases is aCas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also knownas Csn1 and Csx12), Cas100, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2,Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6,Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15,Csf1, Csf2, Csf3, Csf4, or Cpf1 endonuclease; a homolog thereof, arecombination of the naturally occurring molecule thereof,codon-optimized thereof, or modified versions thereof, and combinationsthereof.
 6. The method of claim 5, wherein the method comprisesintroducing into the cell one or more polynucleotides encoding the oneor more DNA endonucleases.
 7. The method of claim 5, wherein the methodcomprises introducing into the cell one or more ribonucleic acids (RNAs)encoding the one or more DNA endonucleases. 8-9. (canceled)
 10. Themethod of claim 1, wherein the method further comprises: introducinginto the cell one or more guide ribonucleic acids (gRNAs).
 11. Themethod of claim 10, wherein the one or more gRNAs are single-moleculeguide RNA (sgRNAs).
 12. (canceled)
 13. The method of claim 10 or 11,wherein the one or more DNA endonucleases is pre-complexed with one ormore gRNAs or one or more sgRNAs.
 14. The method of claim 1, furthercomprising: introducing into the cell a polynucleotide donor templatecomprising at least a portion of the wild-type GUCY2D gene, or cDNA.15.-46. (canceled)
 47. One or more gRNAs for editing a R838H, R838C, orR838S mutation in a GUCY2D gene in a cell from a patient with autosomaldominant Cone-Rod Dystrophy (CORD), the one or more gRNAs comprising aspacer sequence selected from the group consisting of nucleic acidsequences in SEQ ID NOs: 5282-5313, 5398-5409, and 5434-5443 of theSequence Listing.
 48. The one or more gRNAs of claim 47, wherein the oneor more gRNAs are one or more sgRNAs. 49.-53. (canceled)
 54. Atherapeutic for treating a patient with autosomal dominant Cone-RodDystrophy (CORD) formed by a method comprising: introducing one or moreDNA endonucleases; introducing one or more gRNA or one or more sgRNA forediting a R838H, R838C, or R838S mutation in a GUCY2D gene; optionallyintroducing one or more donor template; wherein the one or more gRNAs orsgRNAs comprise a spacer sequence selected from the group consisting ofnucleic acid sequences in SEQ ID NOs: 5282-5313, 5398-5409, 5434-5443 ofthe Sequence Listing. 55.-149. (canceled)